Research Paper
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 of cells themselves to mineral particles while the other is the indirect
mechanism in which microbial metabolites act as bio-reagents
The environmental concerns, and requirements to reduce operating (Govender and Gericke, 2011; Smith and Miettinen, 2006). Various pa-
costs, have provoked substantial research works on introducing innova- rameters such as the chemical or electrostatic interactions, Van der
tive methods or developing current technologies like nding more in- Waals forces, surface free energy components, and the presence of dif-
uential otation and occulation reagents (Lopez et al., 2015; Poorni ferent cations affect the attachment of bio-reagents on mineral surfaces
and Natarajan, 2014). Regarding these vigorous attempts, biological (Lopez et al., 2015; Sharma, 1999). Surfactant characteristics of these bi-
mineral processing approaches are ourishing every day because of ological reagents are due to the presence of functional nonpolar hydro-
being environmentally sustainable, cost effective, and exible carbon chains, and polar carboxyl, hydroxyl, or phosphates species in
(Karthiga and Natarajan, 2015; Shih et al., 2001). the cell wall structure or in the extracellular polymeric substances
The existence and behavior of microorganisms are linked to mineral, (EPS) such as polysaccharides, proteins, glycoproteins or nucleic acids
solution and nutrient properties like particle occulation, dispersion, (Diao et al., 2014; Iqbal and Edyvean, 2004; Poorni and Natarajan,
grain shape and size, ion exchange capability, ionic strength and compo- 2014).
sition of solutions (Dai et al., 2014). So, microbes can be applied as col- Kaolinite and silica are two important industrial minerals which
lectors, activators, depressants, occulants, or dispersants to selectively have vast applications in manufacturing ceramics, porcelain, glass, ce-
separate valuable minerals from their contaminants (Khoshdast et al., ment, paints and cosmetics (Choy et al., 2007). So, kaolinite and silica
2012; Smith and Misra, 1993). This is achieved through selective ad- may be present in industrial efuents that should be removed before
sorption of bacterial cells or their secretions onto mineral surfaces as discharging to the environment (Karthiga and Natarajan, 2015;
biolm, then altering their surface chemistry or making adhesive brid- Yannopoulos et al., 2015). Furthermore, they are usually contaminated
ges between mineral particles (Dubel et al., 1992; Raichur et al., with different impurities, mostly iron (hydr)oxides (Bergaya and
1996). Therefore, two different mechanisms govern the surface bio- Lagaly, 2013). Even silica itself is considered as an impurity in kaolin
treatment. One type is the direct mechanism which involves adhesion clay. Therefore, bio-beneciation which utilizes various types of micro-
organisms like fungi and bacteria (Hosseini and Ahmadi, 2015) is the re-
Corresponding author. cent method of choice. This approach mostly includes bio-otation (Deo
E-mail address: r.hosseini@cc.iut.ac.ir (M.R. Hosseini). 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
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
Intensity
ence of extracellular proteins and polysaccharides of Bacillus subtilis
was investigated by Poorni and Natarajan (2013, 2014). Furthermore,
Q
selective separation of hematite from silica, and kaolin through otation Q Q Q Q
Q Q Q Q
and occulation using Bacillus subtilis and Paenibacillus polymyxa was Q Q Q QQQ Q
examined (Deo and Natarajan, 1998; Sarvamangala and Natarajan, 10 20 30 40 50 60 70 80 90
2011; Shih et al., 2001). 2
Despite the valuable contributions of the above-mentioned litera-
ture to the mineral bio-occulation and bio-separation science, varia- Fig. 1. XRD patterns of (a) kaolin and (b) quartz samples. (K: kaolin, Q: Quartz).
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 2.2. Bacterial growth conditions
together. So, a difference in their bio-occulation behavior can substan-
tially assist their separation. On the other hand, there is no information Lyophilized strain of Bacillus licheniformis (PTCC1320) was pur-
about the occulating ability of B. licheniformis cells. Also, the extracellu- chased from the Iranian Research Organization for Science and Technol-
lar polymeric substances of this bacterium have not been examined in ogy (IROST), Tehran, Iran. Initially, bacterial cells were cultivated on
silica occulation. Therefore, the present study evaluates and compares PDA (potato, 300 g/l; dextrose, 20 g/l; and agar 20 g/l), and also in
the occulation of quartz and kaolin using bacterial cells, extracellular 100 ml liquid pre-culture medium containing peptone, 5 g/l; meat ex-
metabolite, polysaccharide, and proteins of Bacillus licheniformis as tract, 3 g/l; CaCl242H2O, 100 mg/l; MnCl22H2O, 10 mg/l, in a 500 ml
bio-occulant, at various pH values and two levels of mineral Erlenmeyer ask and incubated for 24 h. In order to biosynthesis the
concentrations. required occulants, 10 ml of the pre-culture medium was transferred
to 90 ml of a synthesis medium consisting of glucose, 50 g/l;
2. Materials and methods 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
2.1. Minerals and chemicals 96 h. Incubation was performed at 35 C using Jaltajhiz (JTSL20) orbital
shaker at 150 rpm.
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-
2.3. Preparation of bio-occulant solutions
rity raw quartz sample was supplied by Tooma Minerals Co., Isfahan,
Iran. Both samples were ground and screened, separately using 400
In the present study, bacterial cells, metabolites, puried extracellu-
mesh (38 m) sieves, and the nes accumulated under the sieve were
lar protein and polysaccharide were applied as bio-reagents. To separate
used in all experiments. Also, all the chemical reagents were purchased
bacterial cells and metabolites, the cultures were centrifuged at
from Titrachem, Iran in analytical grade.
5000 rpm, and the precipitated cells were washed with distilled
The X-ray diffraction (XRD) analysis was performed on the screened
water, centrifuged three times, and were used in experiments. The re-
samples using Asenware AW/XDM 300, China in the 2 range of 10
maining liquor itself was used as reagent, and for protein and polysac-
90 (step size: 0.05 2, step time: 1 s, slit width: 0.5) with Cu K radi-
charide extraction. Polysaccharides were extracted from the liquor
ation k = 1.5405 at 40 kV, 30 mA, and illustrated in Fig. 1. According to
according to Manivasagan et al. (2015) by adding cold ethanol to the
the XRD patterns, kaolinite, Al2Si2O5(OH)4, and quartz, SiO2, are the
cell free extract at a volumetric ratio of 2:1. For protein extraction, the
main constituents of kaolin (Fig. 1a), and the quartz sample is almost
procedure previously described by Poorni and Natarajan (2014) was
pure (Fig. 1b). The reections associated with each phase are marked
employed by dissolving ammonium sulfate in the cell free liquor at
and described in Fig. 1. In order to obtain the chemical compositions
4 C to 65% saturation level.
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 2.4. Adsorption evaluation
(LOI). Results are presented in Table 1. Also, specic surface area of the
samples was measured by the Brunauer, Emmett and Teller (BET) Adsorption tests were conducted to compare the tendency of pro-
method of adsorption of N2 gas (Belsorp mini II, Bel, Japan). The temper- tein and polysaccharide ingredients to be adsorbed onto the mineral
ature of the liquid nitrogen was 77 K, and all the samples were particles at different pH values. So, in 50 ml of each solution, containing
outgassed at 150 C overnight under owing nitrogen, before measure- bacteria (~7.2 108 cell/ml), protein or polysaccharide (~72 g/ml), 1 g
ments. According to this analysis, the specic surface areas of the initial of the minerals was suspended. Also, NaCl as an electrolyte was added
quartz and kaolin samples were 0.46 and 4.81 m2/g. Additionally, (10 mM), and pH was adjusted to the levels of 2 to 9. Then, the disper-
their total pore volumes were 6.30 10 3 and 30.66 103 cm3/g, sion was agitated for 15 min on a heater-stirrer at 35 C. Finally, the dif-
respectively. ference between initial and nal concentrations of bacterial cells,
R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188 83
Table 1
Chemical compositions of the kaolin and quartz samples.
Oxide (mass %) SiO2 Al2O3 Fe2O3 TiO2 CaO MgO Na2O K2O L.O.I
Kaolin 63 1 24 1 0.6 0.1 0.04 0.01 1.2 0.2 0.55 0.05 0.4 0.1 0.3 0.1 91
Quartz 98 1 0.2 0.1 0.1 0.1 0.02 0.01 0.14 0.01 0.01 0.01 0.03 0.01 0.06 0.01 NDa
a
ND: Not determined.
protein, and polysaccharide was calculated and reported as the mass of added to 100 ml agitating mineral dispersion (3.6 108 cells/ml) and
reagent (mg) adsorbed onto the unit surface (m2) of mineral powder. the settling test conducted after pH adjustment.
Protein concentration was measured by the Bradford (1976) method,
and phenol-sulfuric acid (Dubois et al., 1956) technique was imple- 2.7. Scanning Electron Microscopy (SEM)
mented for the polysaccharide assessment. In order to identify the bac-
terial adsorption to the minerals, samples were taken from the bacterial SEM images were taken of gold coated quartz and kaolin before and
dispersion before and after treatment with mineral powder. Then the after treatment with bacterial cells, and bacterial protein at pH = 2
bacterial cells were centrifuged (4000 rpm) and transferred to a lysis using scanning electron microscope (SEM, Philips, XI30, The Nether-
buffer containing phosphate buffer saline (PBS) and imidazole, 10 mM. lands) at 20 kV acceleration voltage.
Afterward, the opaque dispersion was sonicated for 2 min (5-s pulses,
amplitude of 20 nm) until becoming clear. After centrifugation, the pro- 3. Results and discussion
tein contents of the remained liquors were measured by Bradford
method. The nal cells and secretions obtained from the bacterial cultures
were applied as bio-occulants in order to investigate the inuences
2.5. Design of experiments of the dispersion pH, solid concentration, and bio-reagent type on the
settling behavior of kaolin and quartz by conducting different bio-oc-
In order to conduct sedimentation experiments, a general factorial culation experiments. Furthermore, the outcomes of the settling tests
design was selected for each mineral. Variables were the type of bio-re- were used in a general factorial design to analyze the signicance and
agent, pH of the sedimentation medium, and mass of the mineral pow- inuence of each parameter on the response variable. Finally, a mathe-
der. The investigated variables and their levels are shown in Table 2. matical model was obtained to predict the bio-occulation behavior of
Each run was done in duplicate, and results were assessed by Design ex- both minerals.
pert 7 software.
3.1. Bio-occulation experiments
2.6. Sedimentation tests
Results of the settling tests on kaolin and quartz using 50 g/l solid
Sedimentation experiments were carried out using different bio-re- concentration are depicted in Fig. 2a and b, respectively. Considering
agents. Therefore, to compare the inuence of the reagent type on the the reagent-free control experiments, kaolin sedimentation with an av-
mineral sedimentation, initially, a control test was conducted without erage of around 30% was generally lower than the mean quartz settling
any additive at the ve specied pH levels. The mineral powder (kaolin percentage (37%) in the examined pH range. According to both dia-
or quartz) was added to 80 ml of deionized water, and its pH was set to grams, increasing the dispersion pH from 1 to 9 reduced the mineral set-
the identied value by dilute sulfuric acid or sodium hydroxide while tling percentage, because at pH values less than the PZC of the minerals,
steering for 5 min. Afterward, the slurry was transferred to a 100 ml which are about 4 and 2 for kaolin and quartz, respectively (Kosmulski,
measuring cylinder and lled with deionized water to exactly 100 ml. 2009), the surface charge is positive and after the point of zero charge it
The cylinder was inverted ten times during 5 min and kept still for becomes progressively negative. Therefore, the repulsive forces be-
3 min. Then, 90 ml of the dispersion was removed and the remained tween the particles caused a reduction in particle settling, although
sediments dried and weighted. Finally, the ratio of the precipitates to this was not obvious for the kaolin sample.
the total powder mass was reported as sedimentation percent. The After addition of the bacterial cells to the mineral dispersions as bio-
same procedure was also implemented when bio-reagents were ap- surfactants, an obvious augmentation in the extent of occulation was
plied. For metabolite, one-fth (20 ml) of the separated metabolite registered. Regarding quartz, this increase was more signicant in acidic
was added to 80 ml of the mineral dispersion during agitation, and solutions of all the applied bio-occulants. Considering kaolin disper-
then pH was adjusted to the set point. For protein and polysaccharide, sions, only 3% improvement in settling was achieved using an acidic en-
each puried component obtained from one culture ask was dissolved vironment in the presence of microorganisms, and changing the pH
in 100 ml deionized water and a similar process was repeated to apply a values from 1 to 9 did not show signicant variation in the amount of
reagent concentration similar to the bacterial metabolite (protein kaolin sedimentation. However, in the case of quartz dispersion, settling
~ 7.2 g/ml, and polysaccharide ~ 0.24 mg/ml). For the bacterial cells, was enhanced by 36% using microbial cells at pH = 1. Increasing the
all the washed precipitated cells from one culture ask, were gradually 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-
Table 2
faces and their ion-exchange properties (Deo and Natarajan, 1998).
Variables and their levels in experimental design.
Mineral surface adheres to the functional groups of the cell wall mostly
Variables Levels due to the attractive electrostatic forces between opposite electric
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 bacteria imparts negative electric charge to their surface (Ahmad
reagent et al., 2011), positively charged amine groups of proteinaceous com-
C Mineral 20 g/l 50 g/l pounds, which are also present on the cell wall, are responsible for the
concentration
binding of quartz and aluminosilicate minerals to gram-positive
84 R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188
45 0.8
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
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- Fig. 3. Adsorption of bio-reagents on (a) kaolin and (b) quartz as a function of pH. (Square,
reagent using slurry concentration of 50 g/l. (square, solid line: cells; circle, dashed line: solid line: cells; Circle, dashed line: protein; Triangle, dotted line: polysaccharide).
protein; triangle, dotted line: polysaccharide; diamond, dash-dotted line: metabolite;
gray solid line: control).
When bacterial culture broth used as bio-reagent, a signicant in-
crease in occulation of the both minerals was observed. Although
bacteria (Deo and Natarajan, 1998). Aluminum as the natural constitu- after the bio-treatment, kaolin settling only increased to 41% at pH =
ent of clay minerals can be hydrolyzed to electrically positive species 5, the greatest improvement in quartz sedimentation percent was
like AlOH+ or Al13(OH32)7+ complexes which act as a bridge between achieved using bacterial metabolite which caused 90% of quartz to be
the two negatively charged groups (Cao et al., 2011; Poorni and settled at pH = 1. Neglecting the nal rise in kaolin sedimentation at
Natarajan, 2013). pH = 9, increasing the dispersion pH generally led to decline in clay set-
The dependence of cell adsorption on the dispersion pH are illustrat- tling percent. At neutral pH, settling of quartz and kaolin reached the
ed in Fig. 3a and b. According to these diagrams, the lower was the pH of value obtained in the control test. Similar results about kaolin-metabo-
the mineral dispersion, the higher was the reagent adsorption. The only lite interaction in acidic (Deo and Natarajan, 1998) and neutral (Poorni
exception was cell adsorption on kaolin particles which culminated at and Natarajan, 2013; Shih et al., 2001) pH ranges were reported in liter-
pH = 7. At this pH, kaolin occulation was also at a maximum. A similar ature. Considering quartz mineral, a signicant reduction in sedimenta-
trend was also reported for kaolin and quartz by Deo and Natarajan tion around neutral pH was reported by Deo and Natarajan (1998), and
(1998), and for quartz by Sarvamangala and Natarajan (2011), while Sarvamangala and Natarajan (2011).
Jiang et al. (2007) indicated a maximum cell adsorption on kaolin at Soil microorganisms excrete extracellular polysaccharide molecules
pH = 3. to their growth environment in order to increase the physical strength
A pH value between 2 and 4 was declared as the PZC of bacterial cells of the soil by making the soil minerals and microstructures bind togeth-
by some researchers (Claessens et al., 2006; Hong and Brown, 2008; er for supporting themselves in water uctuation, and dryness periods
Jiang et al., 2007; Lopez et al., 2015); therefore, in an alkaline solution, (Gomoiu and Catley, 1996). These extracellular polymeric substances
where repulsive forces were dominant between extremely negative (EPS) may be expelled to the culture medium as bacterial metabolites,
surface charges, less adsorption would happen. However, at acidic pH formed onto the cell wall, or adhered on mineral surfaces as biolms.
values around PZC of the minerals and microbial cells, cell-mineral ad- They are made of proteins and polysaccharides synthesized from carbon
hesion was enhanced. Nevertheless, the mentioned increase in the cell and nitrogen sources of the growth medium and contain different func-
adsorption to kaolin particles at neutral to alkaline pH, and subsequent tional groups such as carboxyl, phosphoryl, amide, amino and hydroxyl
improvement in its precipitation (Fig. 2a) implied that other forces like groups (Manivasagan et al., 2015; Poorni and Natarajan, 2014). In the
chemical, Van der Waals or hydrogen bonding might play a role in cell- bio-occulation process, EPS molecules are attached to mineral surfaces,
mineral aggregation (Deo and Natarajan, 1998; Jiang et al., 2007; Lopez inter-bridge the divides between particles and creates steady three-di-
et al., 2015). Regarding Fig. 3a and b, the tendency of kaolin particles for mensional network of ocks which improves the settling rate of mineral
the adsorption of bacterial cells was much lower than quartz, and this particles (Karthiga and Natarajan, 2015).
phenomenon was the reason why the quartz sedimentation was better According to the adsorption graph depicted in Fig. 3a, and b, the
than kaolin after occulation by the cells. Attachment of B. licheniformis overall decrease in precipitation of minerals at neutral to alkaline pH
cells on quartz particles and their occulation effect was discernible in values was associated with the adopted negative charge of metabolite
Fig. 4b. Also, the bacterial cells adhered to the mineral particles were constituents as a result of proton dissociation of carboxyl, phospho-
clearly distinguishable in Fig. 4c. lipids, phosphodiester, hydroxyl and amino groups. This phenomenon
R.A. Ghashoghchi et al. / Applied Clay Science 138 (2017) 8188 85
(a) (b)
(c)
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- at pH = 9 in spite of the repulsive forces could again be attributed to
surfactants (Cao et al., 2011; Deo and Natarajan, 1998). However, in bonding forces other than electrostatics (Jiang et al., 2007; Poorni and
acidic environment, EPS functional species became protonated and ac- Natarajan, 2014). Lin et al. (2016) conrmed that there was a large
quired positive charge which adsorbed negative quartz and clay parti- drop in enthalpy from pH = 5 to 9 for the montmorillonite and kaolinite
cles. The lower sedimentation of kaolin compared to quartz in the pH systems that indicated the adsorption of EPS to clay minerals was less
range of 13 was related to the higher PZC value of kaolin, which caused exothermic at higher pH (Lin et al., 2016). Moreover, the difference in
the surface charge of kaolin to be positive and repulsive against cell free polysaccharide adsorption between Fig. 3a and b, specied that quartz
extracts. Despite the fact that these ndings indicated electrostatic in- was much more inclined to adsorb polysaccharide in comparison to ka-
teractions as important driving forces in EPS adsorption on both min- olin (Deo and Natarajan, 1998). So, the higher occulation efciency of
erals (Cao et al., 2011; Lin et al., 2016; Poorni and Natarajan, 2013, quartz in the presence of bacterial polysaccharides was justied.
2014), a slight increase in polysaccharide and protein adsorption to ka- Considering protein as bio-surfactant, about 78% quartz sedimentation
olin at pH = 9 along with great improvement in its settling behavior was achieved at pH = 1. Further increasing the settling pH resulted in a
compared to pH = 7 suggested that other bonds like Van der Waals sudden drop in quartz occulation at pH = 5 to below the amount regis-
or chemical interactions might attach cell metabolite to kaolin surface. tered in the control test. Generally, as polysaccharide adhesion to quartz
Regarding Fig. 2a, and b, the application of bacterial exo- was more than protein (Fig. 3b) polysaccharide was more inuential in
polysaccharides has led to the lowest settling efciency of two minerals quartz settling, especially at pH = 5. Variations in kaolin settling were sim-
at pH = 7. At this point, the particle sedimentation was even lower than ilar to what was observed for polysaccharide with a minimum point at
what observed in the control test. For quartz, this settling reduction con- pH = 7. The highest kaolin settling percent (about 45%) was obtained at
tinued at higher pH values, but for kaolin particles, another 14% increase pH = 9 using both extracellular proteins and polysaccharides. These out-
was observed at pH = 9. This peculiar uctuation was not specic to comes seem rational if the results of adsorption tests are regarded
exopolysaccharides of B. licheniformis, because it was also previously re- (Fig. 3a). Considering the curves presented in Fig. 3a and b, protein attach-
ported using B. subtilis (Poorni and Natarajan, 2014), and B. nivea ment was prevented at pH values higher than mineral PZC except for ka-
(Gomoiu and Catley, 1996). Additionally, 82% of quartz was settled at olin which showed a high protein adsorption at pH = 9. Although the
pH = 5, which was the highest quartz sedimentation using polysaccha- same observation was reported by some researchers (Cao et al., 2011;
ride among all 5 pH values. Lin et al., 2016), Poorni and Natarajan (2014) presented a completely dif-
Adsorption of polysaccharide to kaolin at different pH values, which ferent pattern of protein and polysaccharide adsorption on kaolin. In their
is illustrated in Fig. 3a, revealed that increasing pH decreased adsorption adsorption curve, EPS adhesion to the clay was at maximum at pH = 7
until pH = 7, but after that there was another increase at pH = 9. This where the lowest extent of settling happened. Also, in contrast to Deo
trend was also shown by Cao et al. (2011), and Lin et al. (2016). So, in- and Natarajan (1998), protein adsorption to quartz was signicantly bet-
crease in kaolin occulation efciency at pH = 9 could be associated ter than kaolin at all the pH ranges (Fig. 3).
with the higher adsorption of polysaccharide at this pH compared to Proteins are comprised of amine and carboxyl groups. At low pH,
pH = 7. Containing COO, OH and PO3 4 groups, exopolysaccharides amine groups are protonated and gain positive charge; on the other
are negatively charged in a wide range of pH values. So, they can interact hand, carboxyl groups of proteins lose hydrogen at high pH, and acquire
with aluminosilicate minerals, and form stable complexes (Deo and negative charge (Ahmad et al., 2011). Therefore, attachment of proteins
Natarajan, 1998). In addition, an acidic environment improves the ex- to the kaolin and quartz surfaces was easier at pH values under 7, in
tracellular polysaccharide adsorption onto the mineral particles, be- which an opposition might exist between surface charges of positive
cause of the positive surface charge of minerals which electrostatically proteins and negative minerals. SEM images of kaolin particles before
attracts negative saccharide polymers. Nevertheless, as repulsive forces and after interaction with proteins are depicted in Fig. 5. Obviously,
rule at high pH values, adsorption declines and mineral occulation de- the micro-sized kaolin akes agglomerated after treatment with bacte-
creases. Augmentation in adsorption to kaolin and settling performance 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 the mineral concentration was 20 g/l, the highest settling difference
in settling efciency. Hence, the applied mineral concentration in all of was 36%, which reached at neutral pH using bacterial cells as bio-surfac-
the experiments was decreased to 20 g/l while the amounts of bio-re- tant, while at 50 g/l mineral concentration, about 4550% difference in
agents remained unchanged. Although, increasing the bio-occulant mineral settling was observed in the pH range of 31, when bacterial
to mineral ratio improves the sedimentation percentage of the minerals, metabolite was applied. So, despite the better occulation efciency at
using high bio-occulant dosages causes oversaturation of bio-surfac- lower mineral concentration, the separation of quartz from kaolin
tants on mineral surface binding sites which reduces particle sedimen- might be more effective at higher mineral concentration.
tation (Karthiga and Natarajan, 2015). The nal consequences are As reported by Cao et al. (2011), Lin et al. (2016), and Poorni and
presented in Fig. 6a and b. Comparing the data presented in this gure Natarajan (2014), protein adsorption to the both mineral surfaces was
and Fig. 2, substantial enhancement in the extent of kaolin occulation higher in comparison to polysaccharide. However, based on the Figs. 2
was achieved compared to previously examined 50 g/l concentration. and 6, adsorption of protein and polysaccharide on the both minerals
Despite this large change in kaolin settling, little improvement in the were pH dependent and affected their occulating performance. Ac-
settling of quartz particles was discerned, especially at high pH values. cording to Fig. 3a, in acidic region, protein adsorption to kaolin was
Using 20 g/l mineral concentration, cell-free extract showed the lower than polysaccharide, but after pH = 6.5, protein was adsorbed
greatest occulating ability among other bio-occulants, which was more. This phenomenon was also reected in the occulation efcien-
83% for kaolin at pH = 3, and 92% for quartz at pH = 1. cies illustrated in Fig. 2a. The same trend was also distinguished for
Considering Figs. 2 and 6, the maximum difference between quartz quartz. At pH = 2 that protein adsorption was higher than polysaccha-
and kaolin settling varied for the two mineral concentrations. When ride (Fig. 3b), quartz sedimentation by protein was also greater (Figs. 2b
and 6b). After this point, polysaccharide adhesion exceeded protein till
90 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-
80 ertheless, since the variation in occulating performance of metabolite
Mass settling percent
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
Table 3 90
ANOVA results for kaolin sedimentation experiments. 80
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