Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Flocculation behaviour of hematite–kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides S. Poorni, K.A. Natarajan ∗ Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 28 August 2013 Accepted 25 September 2013 Available online 18 October 2013 Keywords: Bioflocculation Extracellular proteins Polysaccharides Hematite Kaolinite
a b s t r a c t Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The interaction of clays and iron oxide minerals with biopolymers such as proteins and polysaccharides is of interest to soil chemists and geologists to understand their biostability, mineral cycling, weathering and aggregate formation. Many types of soil microorganisms inhabiting clay and iron oxide mineralized soils and rocks adhere to mineral substrates and form biofilms resulting in secretion of several biopolymers constituting of proteins and polysaccharides, influencing their mineralogy and weathering [1]. Iron cycle in relation to clay mineralogy has been understood with respect to participation of different aerobic and anaerobic microorganisms [2]. Kaolinite formation is reported to be a consequence of bioinduced processes [3]. Iron oxide inclusion in kaolinite matrices is also bioinduced and hence a biological process may be appropriate to remove iron oxides from kaolin clays. Presence of iron in kaolin imparts a brownish colour which limits its industrial applications in ceramic, porcelain, cosmetics and paint manufacturing. Iron removal from clays is thus an important initial step for the production of commercially suitable kaolin containing kaolinite. Physicochemical methods including washing, acid dissolution, froth flotation and electrostatic separation commonly used to bring about iron removal from clays or clay removal from iron ores
∗ Corresponding author. Tel.: +91 80 22932679; fax: +91 80 23600472; Mob: +91 9880250091. E-mail addresses: [email protected] (S. Poorni), [email protected], [email protected] (K.A. Natarajan). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.049
are often inefficient and not environment-friendly. An alternative approach of utilizing microorganisms as surface modifiers to bring about mineral beneficiation has great practical relevance due to its cost effectiveness and environmental acceptability [4]. Under the circumstances, an understanding of consequences interaction of bacterial cells and their extracellular polymeric metabolic products with clay minerals and associated iron oxide will be of great practical significance. Microbially-induced flocculation or dispersion of clay minerals such as kaolinite and iron oxide such as hematite would be useful in their mutual separation and also to achieve supernatant clarity from their suspensions. Since, flocculation–dispersion of mineral fines in an aqueous medium is an interfacial process, it is essential to understand the adsorption and electrokinetic behaviour of bacterial cells and the extracellular polymeric substances in the presence of the above minerals. Use of Bacillus spp., and fungi such as Aspergillus niger to remove iron from low grade kaolin have been reported earlier [5–8]. Iron reducing-microorganisms viz., Bacillus cereus, Bacillus sphaericus, Bacillus pumilus, Mycoides and Pseudomonas mendocina have also been used for the microbial refinement of kaolins [9,10]. However, all such microbial methods so far reported for iron removal from clays are based on reductive dissolution of iron oxides in organic and other acids produced through bacterial or fungal metabolism. The present work assumes great practical significance since it discusses a novel iron oxide-kaolinite separation process based on selective flocculation-dispersion induced by bacterial extracellular proteins and polysaccharides. Bacillus subtilis, a Gram-positive soil microbe was chosen in this work to establish the adsorption behaviour of extracellular
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
polymeric substances such as proteins and polysaccharides on a typical clay mineral, such as kaolinite and iron oxide, such as hematite. Use of Bacillus subtilis in the beneficiation of hematite to remove silica, alumina and calcite has already been reported in Ref. [11]. Bacterial interaction with clays and iron oxides was studied to understand differential bacterial surface affinities towards these minerals which occur closely associated together in nature. Such a study would facilitate development of a bioprocess to beneficiate both iron ores and clay minerals. Biologically-induced selective flocculation of hematite from its mixture with kaolinite was established in this work and its applications in the beneficiation of kaolin clays with respect to iron removal is emphasized. The results of this work are also significant with respect to removal of kaolin from hematitic iron ores. 2. Materials and methods 2.1. Minerals Hand-picked pure mineral samples of hematite and kaolinite were obtained from Alminrock Indscer Fabriks and Indian Bureau of Mines, Bangalore, India, respectively. The minerals were subjected to dry grinding in a porcelain ball mill and sieved to obtain different size fractions. Fractions less than 10 m sizes were obtained through sedimentation. Particle size was determined by Malvern Zetasizer (Nano ZS90) and an average particle size of ∼8–10 m was used for adsorption, flocculation and zeta potential studies. The purity of minerals determined through X-ray diffraction and mineralogical analysis was 99.8% hematite and 98.6% kaolinite. 2.2. Bacterial growth conditions A strain of Bacillus subtilis (NCIM 2655) obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India, was used. Bacterial cells were subcultured in Luria Bertani (LB) medium by inoculating 10 ml of pure strain in 90 ml of LB medium in 250 ml Erlenmeyer flasks and incubated in an Orbitek rotary shaker at 30 ◦ C and 200 rpm [11]. The cell population was estimated using a Petroff-Hausser counting chamber under a Leitz phase contrast microscope (LABORLUX K Wild MPS 12). Bacillus subtilis cells were also grown in the presence of hematite and kaolinite at a pulp density of 10% and adaptation to minerals was considered achieved when the growth rate of adapted strain was identical to that observed in the absence of minerals. Mineralgrown adapted cells were preserved and periodically subcultured in the presence of respective minerals. The mineral samples were also sterilized before interacting with bacterial cultures in order to ensure prevention of contamination from exogenous microorganisms. 2.3. Extraction of extracellular protein (EP) and polysaccharide (ECP) A volume of 1 l solution-grown or minerals-grown bacterial culture was taken and centrifuged at a speed of 8000 rpm for 5 min. The supernatant was filtered through 0.2 m sterile membrane filter paper to obtain cell free extract (CFE). Analytically pure ammonium sulphate was slowly added to a saturation level of 65% with constant stirring at 4 ◦ C. The precipitated protein was dissolved in a minimal volume of 0.1 M Tris–HC1 buffer of pH 7, dialyzed and kept overnight at 4 ◦ C [12]. For ECP extraction, 1 l of two days solution-grown or mineralsgrown bacterial culture was centrifuged and the supernatant filtered through 0.2 m sterile membrane using Millipore vacuum
187
suction pump. The cell free extract was lyophilized in Modulyod 230 freeze dryer at a temperature of −50 ◦ C and a vacuum of 100 millitorr. The solids obtained were dissolved in 10 ml of double distilled water and allowed to cool down to 10 ◦ C, followed by addition of 20 ml of ice cold ethanol to selectively precipitate all the polysaccharide. The precipitate was centrifuged and redissolved in double distilled water followed by ethanol precipitation which was repeated thrice. The precipitated polysaccharide was then dissolved in a minimal volume of double distilled water, dialysed and kept overnight at 4 ◦ C in a refrigerator [13]. 2.4. Isolation and characterization of EP by SDS-PAGE Proteins can be characterized in terms of the molecular size of the constituent polypeptides by SDS-PAGE. Electrophoresis was carried out in vertical position which provides longer separations and the supporting medium was impregnated with buffer solution. The buffer compartments were separated physically to avoid contamination with the products of the electrolysis formed in the electrode compartment. 12% SDS-PAGE gel was used for the purpose and procedures involved are detailed elsewhere [11,12]. 2.5. Adsorption studies For adsorption tests, one gram each of individual mineral sample was suspended in 100 ml of 1 mM KNO3 solution at the desired pH in the presence of known concentration of bacterial extracellular proteins or polysaccharides in 250 ml Erlenmeyer flasks. The suspension was agitated for 15 min on an orbital shaking incubator at 30 ◦ C and 250 rpm and equilibrated at different pH levels. The suspension was then centrifuged at 2000 rpm for 5 min to separate the mineral particles with adsorbed proteins and polysaccharides. The supernatant was further filtered through Whatman 42 filter paper and analyzed for residual protein and polysaccharide [14,15]. 2.6. Zeta potential studies The effect of bacterial interaction on the surface charge of the minerals was studied by Zeta-potential measurements using Malvern Zetasizer (Nano ZS90). For zeta potential studies, 1 g of desired mineral sample was taken in 10−3 M KNO3 and interacted with known amount of EP and ECP at desired pH and period at room temperature. After interaction, mineral particles were separated by filtration and then washed twice or thrice to remove the loosely adsorbed reagents. Zeta potentials of bacterial cells grown in the presence and absence of minerals were also similarly determined as a function of pH. 2.7. Flocculation tests For flocculation tests, 1 g of the desired mineral sample was taken in 100 ml of known bacterial EP and ECP concentration in a measuring cylinder. The cylinder was tumbled 10 times and kept still for 5 min. The supernatant as well as settled products were carefully decanted, filtered and weighed. Selective flocculation tests using 1:1 mineral mixtures were also similarly carried out. 2.8. SEM micrographic analysis SEM studies were carried out using a FEI Sirion, high resolution electron microscope as per procedure described elsewhere [16].
188
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
15
60
a
b
EP from
EP from Solution-grown cells
Hematite-grown cells
12
Kaolinite-grown cells
9 6 3
Adsorption density, µg/g
Adsorption density (µg/g)
Solution-grown cells
0
50
Hematite-grown cells Kaolinite-grown cells
40 30 20 10 0
0
2
4
6
8
10
0
2
4
pH
6
8
10
pH
Fig. 1. Adsorption density of EP secreted by Bacillus subtilis grown in the absence and presence of minerals onto (a) hematite and (b) kaolinite as a function of pH (interaction time = 15 min). (Note: standard deviation varies between ±0.1 and 1.6 for n = 3.)
Adsorption density, µg/g
a
ECP from Solution-grown cells Hematite-grown cells Kaolinite-grown cells
25 20 15 10 5
Adsorption density, µg/g
6
30
b
ECP from Solution-grown cells Hematite-grown cells Kaolinite-grown cells
4
2
0
0 0
2
4
6
8
10
pH
0
2
4
6
8
10
pH
Fig. 2. Adsorption density of ECP secreted by Bacillus subtilis grown in the absence and presence of minerals onto (a) hematite and (b) kaolinite as a function of pH. (Note: standard deviation varies between ±0.1 and 1.0 for n = 3.)
3. Results and discussion Growth curve of Bacillus subtilis in LB medium was initially established. Typical growth curve of Bacillus subtilis in L B medium has been reported elsewhere [11]. Cell growth in presence of hematite and kaolinite was also monitored and mineral-adapted cells were maintained and periodically subcultured in the presence of respective minerals. Extracellular proteins and polysaccharides were extracted from solution- and mineral-grown cells and their metabolites and used for different tests. 3.1. Adsorption behaviour of bacterial EP and ECP Adsorption densities of EP and ECP onto hematite and kaolinite as a function of time at neutral pH were initially established. In general, EP exhibited the highest surface affinity towards kaolinite and the least towards hematite. Maximum surface coverage is attained within about 15 min after which a steady state is reached. EP extracted from kaolinite-grown cells exhibited the highest adsorption density on kaolinite compared to EP extracted from solution-grown and hematite-grown cells. Kaolinite-specific proteins were secreted by the bacterial cells when grown in the presence of kaolinite, the presence of which during bacterial growth promoted enhanced protein secretion. On the other hand, presence of hematite during bacterial growth did not promote
protein generation. For example, SDS-PAGE analysis indicated that kaolinite-specific proteins having molecular weights 20 kDa, 25 kDa, 45 kDa and 50 kDa were secreted only when the cells were grown and adapted to kaolinite. No such mineral-specific proteins were secreted when grown in presence of hematite. Amounts and types of proteins and polysaccharides generated by Bacillus subtilis were influenced by bacterial growth conditions in the presence and absence of the minerals. Adsorption behaviour of extracellular proteins and polysaccharides generated under different growth conditions on hematite and kaolinite was also found to be different. ECP exhibited significantly higher adsorption density onto hematite than on kaolinite. Presence of hematite during bacterial growth resulted in enhanced secretion of polysaccharides which exhibited higher surface affinity towards hematite. Several iron oxidizing bacteria under natural conditions produce exopolysaccharides around cell walls to protect from encrustation [17]. In soil organisms such as B. Subtilis occurring associated with iron oxides, nanocrystals of iron oxides are found to be encrusted in a matrix of polysaccharides. Reactivity of bacterial cells with the extracellular polymers facilitate iron adsorption and solubilisation. Polysaccharides can comples with surface iron atoms and protect the cell. ECP extracted from hematite-grown cells exhibited the highest adsorption density on hematite, followed by ECP extracted from solutionand kaolinite-grown cells.
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
Solution-grown cells Hematite-grown cells Kaolinite-grown cells
20 Zeta potential, mV
Increased cells and polysaccharide adsorption on hematite rendered the mineral surfaces more hydrophilic, while proteinadsorbed kaolinite particles exhibited higher surface hydrophobicity as confirmed by microflotation. Adsorption density of EP and ECP on hematite and kaolinite as a function of pH is illustrated in Figs. 1 and 2. In all cases, adsorption densities of both EP and ECP were found to increase with pH up to about neutral pH, decreasing afterwards. Adsorption of proteins on clays was earlier reported to be pH dependent reaching a maximum close to the IEP [18]. Also, the solubility of these biopolymers is influenced by pH. Growth of Bacillus subtilis is accompanied by production of extracellular polymeric substances which are bound to the cell surfaces, released into metabolites and also associated with biofilms formed on mineral surfaces. Such extracellular products are a heterogeneous mixture of proteins and polysaccharides, containing various-functional groups like carboxyl, phosphoryl, amide, amino and hydroxyl. Adsorption of the various proteinaceous and polysaccharide groups onto different minerals is governed by various forces such as electrostatic, hydrophobic and polymer-polymer interactions [1]. From the above results, it becomes clear that proteinaceous constituents were preferentially adsorbed on kaolinite. Polysaccharide constituents containing OH, COOH and phosphoryl groups were significantly adsorbed on hydroxylated hematite surfaces. The observed decrease of protein and polysaccaharide adsorption with increasing pH may be attributed to protonation of EPS groups (amino and hydroxyl), which occurs at a lower pH of about 3 and the positively charged cationic groups can adsorb on negatively charged clays. Similarly, negatively charged polysaccharide and phosphoryl functional groups (COOH, OH, PO4 2− ) can adsorb on positively charged hematite surface at pH lower than IEP (about 6), such adsorption decreasing at pH values higher than 7 [1]. Electrostatic forces play a role in protein and polysaccharide adsorption on kaolinite and hematite whose IEPs were located at pH of 2.1 and 5.5, respectively. Cationic functional groups of proteins will increasingly adsorb on negatively charged kaolinite surfaces, while anionic polysaccharide (OH, COOH) and phosphoryl groups will be increasingly adsorbed on positively charged hydroxylated hematite at pH lower than about 6. Adsorption density of all types of EPs extracted from cells-grown under different conditions was found to be the higher on kaolinite, while the lowest on hematite. Almost 3-4 times higher amount of EP were found to be adsorbed on kaolinite compared to hematite. EP isolated from kaolinite-grown cells and their metabolites exhibited the highest adsorption on kaolinite, whereas cells growth on kaolinite decreased EP adsorption on hematite substantially. Similarly, EP from hematite-grown cells exhibited lower surface affinity towards kaolinite. Adsorption density of ECP isolated from bacterial cells and metabolites, irrespective of the type of growth conditions, was the highest on hematite and the lowest on kaolinite. ECP from hematite-grown cells exhibited significantly higher adsorption on hematite than on kaolinite. It becomes very clear from the above results that mineralspecificity for cells as well as their extracellular products can be developed through bacterial growth and subsequent adaptation in the presence of desired minerals. For example, bacterial cells which were grown in the presence of hematite were found to adhere profusely on hematite-surfaces. Adhesion of solution- and kaolinite-grown cells on kaolinite was lower compared to that on hematite. Also, bacterial cells which were grown and adapted to hematite and kaolinite exhibited changes in morphological features when compared to solution-grown cells. Confirmatory adsorption tests using BSA and starch as standard protein and polysaccharide were also carried out. Both BSA and starch exhibited strong pH dependence on their adsorption to
189
0 0
2
4
6
8
10
-20
-40
pH Fig. 3. Zeta potential of cells grown in the absence and presence of minerals as a function of pH. (Note: standard deviation varies between ±1.0 and 2.5 for n = 5.)
hematite and kaolinite, similar to that observed in the presence of EP and ECP extracted from bacterial metabolites. BSA protein exhibited the highest adsorption density only on kaolinite while starch adsorption was the highest on hematite. 3.2. Electrokinetic behaviour of bacterial cells and minerals Bacteria-mineral interactions result in significant surface chemical changes on both cell surfaces and interacted minerals. Zeta potential changes with pH for bacterial cells before and after interaction with hematite and kaolinite are shown in Fig. 3. Before mineral interaction, IEP of Bacillus subtilis was located at a pH of about 2.5. Shifts in IEP and zeta potentials for bacterial cells which were grown in the presence of hematite and kaolinite are also shown. IEP of hematite-grown cells is seen to be shifted to a higher pH value of about 4.5 presumably due to influence of adsorbed iron from hematite during growth. Hematite-grown cells when subjected to prolonged growth (adaptation) in the presence of hematite exhibited further IEP shift towards a lower pH of about 2.0 (not shown in figure), presumably due to enhanced bacterial secretion of polysaccharides on cell walls and its adsorption onto hematite. Similarly, kaolinite-grown cells after prolonged exposure to kaolinite exhibited an IEP shift to pH of about 3.5 (not shown in the figure). Zeta potential and IEP changes exhibited by bacterial cells are due to alteration in cell wall composition of the biopolymers brought about by interaction and growth in the presence of hematite and kaolinite. Growth and adaptation of cells in the presence of kaolinite promoted enhanced generation of newer proteinaceous groups, while the presence of hematite resulted in higher secretion of extracellular polysaccharides. For example, amount of ECP secreted by Bacillus subtilis when grown in the presence of hematite was twice that produced in its absence. Similarly, kaolinite-grown cells were observed to secrete significantly higher amount of proteins (about 90 mg/l) compared to only 40 mg/l in the presence of hematite. Similarly, higher adsorption of proteins onto kaolinite and of polysaccharides on hematite will bring about significant changes in the electrokinetic behaviour of the minerals, as illustrated in Figs. 4 and 5. Electrokinetic changes on hematite and kaolinite brought about by interaction with mineral-adapted cells and their extracellular products were studied. The following observations are noteworthy: a. Significant negative shifts in hematite IEP could be observed after interaction with hematite- and kaolinite-grown cells. b. IEP of kaolinite shifted to higher pH (2.1 to 3–3.5) after similar interactions.
190
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
15
Hematite before interaction Interacted with EP from hematite-grown cells Interacted with EP from kaolinite-grown cells
30
0
0 0
2
4
6
8
10
-15 -30 -45
a
Zeta potential, mV
15 Zeta potential, mV
Kaolinite before interaction Interacted with EP from hematite-grown cells Interacted with EP from kaolinite-grown cells
0
4
6
8
10
-15
-30
a
-45
pH
2
pH
Fig. 4. Zeta potential of (a) hematite and (b) kaolinite before and after interaction with EP secreted by Bacillus subtilis grown in the presence of minerals as a function of pH. (Note: standard deviation varies between ±0.8 and 2.4 for n = 5.)
30
15
Hematite before interaction
Kaolinite before interaction
Interacted with ECP from hematite-grown cells
Interacted with ECP from hematite-grown cells
Interacted with ECP from kaolinite-grown cells
Interacted with ECP from kaolinite-grown cells
0 0
2
4
6
8
10
-15
-30
a
Zeta potential, mV
Zeta potential, mV
15
0 0
4
6
8
10
-15
-30
-45 pH
2
b pH
Fig. 5. Zeta potential of (a) hematite and (b) kaolinite before and after interaction with ECP secreted by Bacillus subtilis grown in the presence of minerals as a function of pH. (Note: standard deviation varies between ±0.2 and 2.5 for n = 5.)
c. Such IEP shifts observed with hematite and kaolinite are due to bacterial secretion and subsequent adsorption of EP and ECP onto minerals. d. Significant surface adsorption of EP on kaolinite resulted in IEP shift of kaolinite from pH 2.1 to 4.0. Interaction with EP extracted from mineral-grown cell free culture shifted the IEP of hematite from pH 5.5 to about 4.0 e. IEP’s of hematite and kaolinite were shifted to lower pH values with zeta potentials becoming more negative after interaction with ECP extracted from mineral-grown cells. Effect of mineral interaction with extracellular proteins and polysaccharides on the shifts in IEP’s and zeta potentials of hematite and kaolinite was cross checked using BSA and starch as standard proteins and polysaccharide and similar results were obtained. 3.3. Flocculation–dispersion behaviour of hematite and kaolinite Flocculation-dispersion behaviour of hematite and kaolinite particles before and after interaction with EP and ECP extracted from bacterial cells under different growth conditions was then established as shown in Figs. 6 and 7. In the absence of bacterial interactions, settling rate of both hematite and kaolinite decreased sharply with increase in pH. Corresponding to neutral pH conditions, maximum settling (flocculation) for hematite was achieved using ECP extracted from hematite-grown cells
followed by ECP from kaolinite-grown cells. Similarly, maximum dispersion of kaolinite was observed after interaction with EP extracted from either kaolinite- or solution-grown cells at neutral pH. EP extracted from hematite-grown cells did not promote higher kaolinite dispersion. On the other hand, ECP extracted from hematite- and kaolinitegrown cells was also effective in bringing significant dispersion of kaolinite particles. Higher-surface affinity of kaolinite for EP and of ECP for hematite is responsible for the above observed flocculationdispersion behaviour. Flocculation-dispersion of hematite–kaolinite mixtures using different biotreatments was studied in detail and typical results are illustrated in Table 1. Pretreatment with EP extracted from bacterial cells and their metabolites after growth in the presence of hematite brought about significant flocculation of hematite and dispersion of kaolinite. Similar flocculation of hematite as well as kaolinite dispersion could be achieved after interaction with ECP isolated from kaoliniteand hematite-grown cells and their metabolites. Through prior biotreatments with such extracellular polymeric substances, effective selective flocculation of hematite from its mixture with kaolinite could be achieved. The above observations open up newer possibilities in developing a cost-effective, energy-efficient and environment-friendly bioprocess for removal of iron oxides (hematite) from kaolin and china clays. To demonstrate the new bioprocess, iron removal
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
120
120
a
b
Hematite before interaction
Kaolinite before interaction Interacted with EP from
Interacted with EP from
100
Solution-grown cells
Solution-grown cells
Hematite-grown cells
Hematite-grown cells
Kaolinite-grown cells
Kaolinite-grown cells
Percent settled
Percent settled
100
191
80 60 40
80 60 40 20
20
0
0 0
2
4
6
8
0
10
2
4
pH
6
8
10
pH
Fig. 6. Flocculation behaviour of (a) hematite and (b) kaolinite before and after interaction with different bacterial extracellular proteins (EP) as a function of pH. (Note: standard deviation varies between ±1.0 and 4.0 for n = 5.)
140
140 a
Interacted with ECP from
Kaolinite before interaction Interacted with ECP from
120
Solution-grown cells
120
Solution-grown cells
Hematite-grown cells
Hematite-grown cells
Kaolinite-grown cells
100
Percent settled
Percent settled
b
Hematite before interaction
80 60 40
100
Kaolinite-grown cells
80 60 40 20
20
0
0 0
2
4
6
8
0
10
2
4
6
8
10
pH
pH
Fig. 7. Flocculation behaviour of (a) hematite and (b) kaolinite before and after interaction with different bacterial extracellular polysaccharides (ECP) as a function of pH. (Note: standard deviation varies between ±2.0 and 4.0 for n = 5.) Table 1 Flocculation–dispersion of hematite and kaolinite after different biotreatments (note: standard deviation varies between ±1.0 and 3.5 for n=5). Type of biotreatment
Control (no biotreatment) EP from kaolinite-grown cells and metabolites ECP from hematite-grown cells and metabolites ECP from kaolinite-grown cells and metabolites
Percent weight settled in 5 min 1:1 Mixture
Kaolinite only
Hematite only
Kaolinite
Hematite
45 08
60 80
50 10
65 85
05
98
15
95
10
90
20
80
studies were carried out using kaolin clay samples containing up to 2% iron as Fe2 O3 . After 5–10 min interaction with hematite-grown cells of Bacillus subtilis and ECP extracted from the metabolites, up to 80–90% of iron could be removed from the kaolin clay sample through selective flocculation. The developed bioprocess could be compared with existing chemical alternatives such as selective flocculation and flotation using toxic chemicals such as amines and polyacrylamides. Efficient separation of kaolin clays from iron oxides or of iron oxides from clays is not possible through
such methods including high intensity magnetic separation and selective flocculation–dispersion. A bioprocess, on the otherhand, would be more process efficient, cost effective, energy efficient and environment–friendly. 4. Conclusions Bacillus subtilis was grown and adapted in the presence of hematite and kaolinite. Kaolinite-grown (adapted) cells secreted
192
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
enhanced quantities of mineral-specific proteins and cells grown in the presence of hematite promoted higher secretion of polysaccharides. Extracellular proteins (EP) exhibited higher surface affinity towards kaolinite, which was rendered hydrophobic. Increased adsorption of exopolysaccharides (ECP) onto hematite resulted in enhanced surface hydrophilicity. Significant surface changes in terms of shifts in IEPs and zeta potentials were observed both on bacterial cells and minerals after interaction with EP and ECP. Flocculation of hematite particles in an aqueous medium was found to be promoted after interaction with ECP from mineral-grown cells and their metabolites, while kaolinite was efficiently dispersed after such interactions. Interaction with EP from kaolinite-grown cells also promoted dispersion of kaolinite. Iron oxides such as Fe2 O3 (hematite) which occurs as impurities in kaolin clays can be efficiently removed through prior interaction with EP and ECP from mineral-grown Bacillus subtilis through selective bioflocculation of hematite and dispersion of kaolinite at neutral pH.
Academy of Sciences (India) for Platinum Jubilee Senior Scientist Fellowship to the corresponding author (Prof. K.A. Natarajan). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
Acknowledgements The authors are thankful to the Department of Science and Technology, Government of India, New Delhi for providing financial support to this research work. Thanks are also due to the National
[14] [15] [16] [17] [18]
Y. Cao, X. Wei, P. Cai, Q. Huang, X. Rong, W. Liang, Colloid. Surf. B 83 (2011) 122. E.S. Shelobolina, S.M. Pickering, D.R. Lovely, Clays Clay Miner. 53 (2005) 580. S. Fiore, S. Dumontet, F.J. Huertas, V. Pasquale, Appl. Clay Sci. 53 (2011) 566. L.M.S. de Mesquita, F.F. Lins, M.L. Turem, Int. J. Miner. Process. 71 (2003) 31. I. Styriakova, I. Styriak, Ceramics—Silikaty 44 (2000) 135. C. Cameselle, M.J. Nunez, J.M. Lema, J. Chem. Technol. Biotechnol. 70 (1997) 349. S.K. Mandal, P.C. Banerjee, Int. J. Miner. Process. 74 (2004) 263. M.R. Hosseini, M. Pazouki, M. Ranjbar, M. Habibian, Appl. Clay Sci. 37 (2007) 251. J.E. Kostka, E. Haefele, R. Viehweger, J.W. Stucki, Environ. Sci. Technol. 33 (1999) 3127. K.S. Cho, E.Y. Lee, H.W. Ryu, Appl. Clay Sci. 22 (2002) 47. H.Sarvamangala, K.A. Natarajan, Int. J. Miner. Process. 99 (2011) 70. M.P. Deutscher, Methods in Enzymology Guide to Protein Purification, Academic Press, New York, 1990. D.T. Plummer, An Introduction to Practical Biochemistry, 2, McGraw-Hill, London, 1978. M.M. Bradford, Anal. Biochem. 72 (1976) 248. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350. M.R. Sabari Prakasan, K.A. Natarajan, Colloids Surf., B 78 (2010) 163. D. Fortin, S. Langley, Earth-Sci. Rev. 72 (2005) 1. B.K.G. Theng, Clays Clay Miner. 30 (1982) 1.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Flocculation behaviour of hematite–kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides S. Poorni, K.A. Natarajan ∗ Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 28 August 2013 Accepted 25 September 2013 Available online 18 October 2013 Keywords: Bioflocculation Extracellular proteins Polysaccharides Hematite Kaolinite
a b s t r a c t Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The interaction of clays and iron oxide minerals with biopolymers such as proteins and polysaccharides is of interest to soil chemists and geologists to understand their biostability, mineral cycling, weathering and aggregate formation. Many types of soil microorganisms inhabiting clay and iron oxide mineralized soils and rocks adhere to mineral substrates and form biofilms resulting in secretion of several biopolymers constituting of proteins and polysaccharides, influencing their mineralogy and weathering [1]. Iron cycle in relation to clay mineralogy has been understood with respect to participation of different aerobic and anaerobic microorganisms [2]. Kaolinite formation is reported to be a consequence of bioinduced processes [3]. Iron oxide inclusion in kaolinite matrices is also bioinduced and hence a biological process may be appropriate to remove iron oxides from kaolin clays. Presence of iron in kaolin imparts a brownish colour which limits its industrial applications in ceramic, porcelain, cosmetics and paint manufacturing. Iron removal from clays is thus an important initial step for the production of commercially suitable kaolin containing kaolinite. Physicochemical methods including washing, acid dissolution, froth flotation and electrostatic separation commonly used to bring about iron removal from clays or clay removal from iron ores
∗ Corresponding author. Tel.: +91 80 22932679; fax: +91 80 23600472; Mob: +91 9880250091. E-mail addresses: [email protected] (S. Poorni), [email protected], [email protected] (K.A. Natarajan). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.049
are often inefficient and not environment-friendly. An alternative approach of utilizing microorganisms as surface modifiers to bring about mineral beneficiation has great practical relevance due to its cost effectiveness and environmental acceptability [4]. Under the circumstances, an understanding of consequences interaction of bacterial cells and their extracellular polymeric metabolic products with clay minerals and associated iron oxide will be of great practical significance. Microbially-induced flocculation or dispersion of clay minerals such as kaolinite and iron oxide such as hematite would be useful in their mutual separation and also to achieve supernatant clarity from their suspensions. Since, flocculation–dispersion of mineral fines in an aqueous medium is an interfacial process, it is essential to understand the adsorption and electrokinetic behaviour of bacterial cells and the extracellular polymeric substances in the presence of the above minerals. Use of Bacillus spp., and fungi such as Aspergillus niger to remove iron from low grade kaolin have been reported earlier [5–8]. Iron reducing-microorganisms viz., Bacillus cereus, Bacillus sphaericus, Bacillus pumilus, Mycoides and Pseudomonas mendocina have also been used for the microbial refinement of kaolins [9,10]. However, all such microbial methods so far reported for iron removal from clays are based on reductive dissolution of iron oxides in organic and other acids produced through bacterial or fungal metabolism. The present work assumes great practical significance since it discusses a novel iron oxide-kaolinite separation process based on selective flocculation-dispersion induced by bacterial extracellular proteins and polysaccharides. Bacillus subtilis, a Gram-positive soil microbe was chosen in this work to establish the adsorption behaviour of extracellular
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
polymeric substances such as proteins and polysaccharides on a typical clay mineral, such as kaolinite and iron oxide, such as hematite. Use of Bacillus subtilis in the beneficiation of hematite to remove silica, alumina and calcite has already been reported in Ref. [11]. Bacterial interaction with clays and iron oxides was studied to understand differential bacterial surface affinities towards these minerals which occur closely associated together in nature. Such a study would facilitate development of a bioprocess to beneficiate both iron ores and clay minerals. Biologically-induced selective flocculation of hematite from its mixture with kaolinite was established in this work and its applications in the beneficiation of kaolin clays with respect to iron removal is emphasized. The results of this work are also significant with respect to removal of kaolin from hematitic iron ores. 2. Materials and methods 2.1. Minerals Hand-picked pure mineral samples of hematite and kaolinite were obtained from Alminrock Indscer Fabriks and Indian Bureau of Mines, Bangalore, India, respectively. The minerals were subjected to dry grinding in a porcelain ball mill and sieved to obtain different size fractions. Fractions less than 10 m sizes were obtained through sedimentation. Particle size was determined by Malvern Zetasizer (Nano ZS90) and an average particle size of ∼8–10 m was used for adsorption, flocculation and zeta potential studies. The purity of minerals determined through X-ray diffraction and mineralogical analysis was 99.8% hematite and 98.6% kaolinite. 2.2. Bacterial growth conditions A strain of Bacillus subtilis (NCIM 2655) obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India, was used. Bacterial cells were subcultured in Luria Bertani (LB) medium by inoculating 10 ml of pure strain in 90 ml of LB medium in 250 ml Erlenmeyer flasks and incubated in an Orbitek rotary shaker at 30 ◦ C and 200 rpm [11]. The cell population was estimated using a Petroff-Hausser counting chamber under a Leitz phase contrast microscope (LABORLUX K Wild MPS 12). Bacillus subtilis cells were also grown in the presence of hematite and kaolinite at a pulp density of 10% and adaptation to minerals was considered achieved when the growth rate of adapted strain was identical to that observed in the absence of minerals. Mineralgrown adapted cells were preserved and periodically subcultured in the presence of respective minerals. The mineral samples were also sterilized before interacting with bacterial cultures in order to ensure prevention of contamination from exogenous microorganisms. 2.3. Extraction of extracellular protein (EP) and polysaccharide (ECP) A volume of 1 l solution-grown or minerals-grown bacterial culture was taken and centrifuged at a speed of 8000 rpm for 5 min. The supernatant was filtered through 0.2 m sterile membrane filter paper to obtain cell free extract (CFE). Analytically pure ammonium sulphate was slowly added to a saturation level of 65% with constant stirring at 4 ◦ C. The precipitated protein was dissolved in a minimal volume of 0.1 M Tris–HC1 buffer of pH 7, dialyzed and kept overnight at 4 ◦ C [12]. For ECP extraction, 1 l of two days solution-grown or mineralsgrown bacterial culture was centrifuged and the supernatant filtered through 0.2 m sterile membrane using Millipore vacuum
187
suction pump. The cell free extract was lyophilized in Modulyod 230 freeze dryer at a temperature of −50 ◦ C and a vacuum of 100 millitorr. The solids obtained were dissolved in 10 ml of double distilled water and allowed to cool down to 10 ◦ C, followed by addition of 20 ml of ice cold ethanol to selectively precipitate all the polysaccharide. The precipitate was centrifuged and redissolved in double distilled water followed by ethanol precipitation which was repeated thrice. The precipitated polysaccharide was then dissolved in a minimal volume of double distilled water, dialysed and kept overnight at 4 ◦ C in a refrigerator [13]. 2.4. Isolation and characterization of EP by SDS-PAGE Proteins can be characterized in terms of the molecular size of the constituent polypeptides by SDS-PAGE. Electrophoresis was carried out in vertical position which provides longer separations and the supporting medium was impregnated with buffer solution. The buffer compartments were separated physically to avoid contamination with the products of the electrolysis formed in the electrode compartment. 12% SDS-PAGE gel was used for the purpose and procedures involved are detailed elsewhere [11,12]. 2.5. Adsorption studies For adsorption tests, one gram each of individual mineral sample was suspended in 100 ml of 1 mM KNO3 solution at the desired pH in the presence of known concentration of bacterial extracellular proteins or polysaccharides in 250 ml Erlenmeyer flasks. The suspension was agitated for 15 min on an orbital shaking incubator at 30 ◦ C and 250 rpm and equilibrated at different pH levels. The suspension was then centrifuged at 2000 rpm for 5 min to separate the mineral particles with adsorbed proteins and polysaccharides. The supernatant was further filtered through Whatman 42 filter paper and analyzed for residual protein and polysaccharide [14,15]. 2.6. Zeta potential studies The effect of bacterial interaction on the surface charge of the minerals was studied by Zeta-potential measurements using Malvern Zetasizer (Nano ZS90). For zeta potential studies, 1 g of desired mineral sample was taken in 10−3 M KNO3 and interacted with known amount of EP and ECP at desired pH and period at room temperature. After interaction, mineral particles were separated by filtration and then washed twice or thrice to remove the loosely adsorbed reagents. Zeta potentials of bacterial cells grown in the presence and absence of minerals were also similarly determined as a function of pH. 2.7. Flocculation tests For flocculation tests, 1 g of the desired mineral sample was taken in 100 ml of known bacterial EP and ECP concentration in a measuring cylinder. The cylinder was tumbled 10 times and kept still for 5 min. The supernatant as well as settled products were carefully decanted, filtered and weighed. Selective flocculation tests using 1:1 mineral mixtures were also similarly carried out. 2.8. SEM micrographic analysis SEM studies were carried out using a FEI Sirion, high resolution electron microscope as per procedure described elsewhere [16].
188
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
15
60
a
b
EP from
EP from Solution-grown cells
Hematite-grown cells
12
Kaolinite-grown cells
9 6 3
Adsorption density, µg/g
Adsorption density (µg/g)
Solution-grown cells
0
50
Hematite-grown cells Kaolinite-grown cells
40 30 20 10 0
0
2
4
6
8
10
0
2
4
pH
6
8
10
pH
Fig. 1. Adsorption density of EP secreted by Bacillus subtilis grown in the absence and presence of minerals onto (a) hematite and (b) kaolinite as a function of pH (interaction time = 15 min). (Note: standard deviation varies between ±0.1 and 1.6 for n = 3.)
Adsorption density, µg/g
a
ECP from Solution-grown cells Hematite-grown cells Kaolinite-grown cells
25 20 15 10 5
Adsorption density, µg/g
6
30
b
ECP from Solution-grown cells Hematite-grown cells Kaolinite-grown cells
4
2
0
0 0
2
4
6
8
10
pH
0
2
4
6
8
10
pH
Fig. 2. Adsorption density of ECP secreted by Bacillus subtilis grown in the absence and presence of minerals onto (a) hematite and (b) kaolinite as a function of pH. (Note: standard deviation varies between ±0.1 and 1.0 for n = 3.)
3. Results and discussion Growth curve of Bacillus subtilis in LB medium was initially established. Typical growth curve of Bacillus subtilis in L B medium has been reported elsewhere [11]. Cell growth in presence of hematite and kaolinite was also monitored and mineral-adapted cells were maintained and periodically subcultured in the presence of respective minerals. Extracellular proteins and polysaccharides were extracted from solution- and mineral-grown cells and their metabolites and used for different tests. 3.1. Adsorption behaviour of bacterial EP and ECP Adsorption densities of EP and ECP onto hematite and kaolinite as a function of time at neutral pH were initially established. In general, EP exhibited the highest surface affinity towards kaolinite and the least towards hematite. Maximum surface coverage is attained within about 15 min after which a steady state is reached. EP extracted from kaolinite-grown cells exhibited the highest adsorption density on kaolinite compared to EP extracted from solution-grown and hematite-grown cells. Kaolinite-specific proteins were secreted by the bacterial cells when grown in the presence of kaolinite, the presence of which during bacterial growth promoted enhanced protein secretion. On the other hand, presence of hematite during bacterial growth did not promote
protein generation. For example, SDS-PAGE analysis indicated that kaolinite-specific proteins having molecular weights 20 kDa, 25 kDa, 45 kDa and 50 kDa were secreted only when the cells were grown and adapted to kaolinite. No such mineral-specific proteins were secreted when grown in presence of hematite. Amounts and types of proteins and polysaccharides generated by Bacillus subtilis were influenced by bacterial growth conditions in the presence and absence of the minerals. Adsorption behaviour of extracellular proteins and polysaccharides generated under different growth conditions on hematite and kaolinite was also found to be different. ECP exhibited significantly higher adsorption density onto hematite than on kaolinite. Presence of hematite during bacterial growth resulted in enhanced secretion of polysaccharides which exhibited higher surface affinity towards hematite. Several iron oxidizing bacteria under natural conditions produce exopolysaccharides around cell walls to protect from encrustation [17]. In soil organisms such as B. Subtilis occurring associated with iron oxides, nanocrystals of iron oxides are found to be encrusted in a matrix of polysaccharides. Reactivity of bacterial cells with the extracellular polymers facilitate iron adsorption and solubilisation. Polysaccharides can comples with surface iron atoms and protect the cell. ECP extracted from hematite-grown cells exhibited the highest adsorption density on hematite, followed by ECP extracted from solutionand kaolinite-grown cells.
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
Solution-grown cells Hematite-grown cells Kaolinite-grown cells
20 Zeta potential, mV
Increased cells and polysaccharide adsorption on hematite rendered the mineral surfaces more hydrophilic, while proteinadsorbed kaolinite particles exhibited higher surface hydrophobicity as confirmed by microflotation. Adsorption density of EP and ECP on hematite and kaolinite as a function of pH is illustrated in Figs. 1 and 2. In all cases, adsorption densities of both EP and ECP were found to increase with pH up to about neutral pH, decreasing afterwards. Adsorption of proteins on clays was earlier reported to be pH dependent reaching a maximum close to the IEP [18]. Also, the solubility of these biopolymers is influenced by pH. Growth of Bacillus subtilis is accompanied by production of extracellular polymeric substances which are bound to the cell surfaces, released into metabolites and also associated with biofilms formed on mineral surfaces. Such extracellular products are a heterogeneous mixture of proteins and polysaccharides, containing various-functional groups like carboxyl, phosphoryl, amide, amino and hydroxyl. Adsorption of the various proteinaceous and polysaccharide groups onto different minerals is governed by various forces such as electrostatic, hydrophobic and polymer-polymer interactions [1]. From the above results, it becomes clear that proteinaceous constituents were preferentially adsorbed on kaolinite. Polysaccharide constituents containing OH, COOH and phosphoryl groups were significantly adsorbed on hydroxylated hematite surfaces. The observed decrease of protein and polysaccaharide adsorption with increasing pH may be attributed to protonation of EPS groups (amino and hydroxyl), which occurs at a lower pH of about 3 and the positively charged cationic groups can adsorb on negatively charged clays. Similarly, negatively charged polysaccharide and phosphoryl functional groups (COOH, OH, PO4 2− ) can adsorb on positively charged hematite surface at pH lower than IEP (about 6), such adsorption decreasing at pH values higher than 7 [1]. Electrostatic forces play a role in protein and polysaccharide adsorption on kaolinite and hematite whose IEPs were located at pH of 2.1 and 5.5, respectively. Cationic functional groups of proteins will increasingly adsorb on negatively charged kaolinite surfaces, while anionic polysaccharide (OH, COOH) and phosphoryl groups will be increasingly adsorbed on positively charged hydroxylated hematite at pH lower than about 6. Adsorption density of all types of EPs extracted from cells-grown under different conditions was found to be the higher on kaolinite, while the lowest on hematite. Almost 3-4 times higher amount of EP were found to be adsorbed on kaolinite compared to hematite. EP isolated from kaolinite-grown cells and their metabolites exhibited the highest adsorption on kaolinite, whereas cells growth on kaolinite decreased EP adsorption on hematite substantially. Similarly, EP from hematite-grown cells exhibited lower surface affinity towards kaolinite. Adsorption density of ECP isolated from bacterial cells and metabolites, irrespective of the type of growth conditions, was the highest on hematite and the lowest on kaolinite. ECP from hematite-grown cells exhibited significantly higher adsorption on hematite than on kaolinite. It becomes very clear from the above results that mineralspecificity for cells as well as their extracellular products can be developed through bacterial growth and subsequent adaptation in the presence of desired minerals. For example, bacterial cells which were grown in the presence of hematite were found to adhere profusely on hematite-surfaces. Adhesion of solution- and kaolinite-grown cells on kaolinite was lower compared to that on hematite. Also, bacterial cells which were grown and adapted to hematite and kaolinite exhibited changes in morphological features when compared to solution-grown cells. Confirmatory adsorption tests using BSA and starch as standard protein and polysaccharide were also carried out. Both BSA and starch exhibited strong pH dependence on their adsorption to
189
0 0
2
4
6
8
10
-20
-40
pH Fig. 3. Zeta potential of cells grown in the absence and presence of minerals as a function of pH. (Note: standard deviation varies between ±1.0 and 2.5 for n = 5.)
hematite and kaolinite, similar to that observed in the presence of EP and ECP extracted from bacterial metabolites. BSA protein exhibited the highest adsorption density only on kaolinite while starch adsorption was the highest on hematite. 3.2. Electrokinetic behaviour of bacterial cells and minerals Bacteria-mineral interactions result in significant surface chemical changes on both cell surfaces and interacted minerals. Zeta potential changes with pH for bacterial cells before and after interaction with hematite and kaolinite are shown in Fig. 3. Before mineral interaction, IEP of Bacillus subtilis was located at a pH of about 2.5. Shifts in IEP and zeta potentials for bacterial cells which were grown in the presence of hematite and kaolinite are also shown. IEP of hematite-grown cells is seen to be shifted to a higher pH value of about 4.5 presumably due to influence of adsorbed iron from hematite during growth. Hematite-grown cells when subjected to prolonged growth (adaptation) in the presence of hematite exhibited further IEP shift towards a lower pH of about 2.0 (not shown in figure), presumably due to enhanced bacterial secretion of polysaccharides on cell walls and its adsorption onto hematite. Similarly, kaolinite-grown cells after prolonged exposure to kaolinite exhibited an IEP shift to pH of about 3.5 (not shown in the figure). Zeta potential and IEP changes exhibited by bacterial cells are due to alteration in cell wall composition of the biopolymers brought about by interaction and growth in the presence of hematite and kaolinite. Growth and adaptation of cells in the presence of kaolinite promoted enhanced generation of newer proteinaceous groups, while the presence of hematite resulted in higher secretion of extracellular polysaccharides. For example, amount of ECP secreted by Bacillus subtilis when grown in the presence of hematite was twice that produced in its absence. Similarly, kaolinite-grown cells were observed to secrete significantly higher amount of proteins (about 90 mg/l) compared to only 40 mg/l in the presence of hematite. Similarly, higher adsorption of proteins onto kaolinite and of polysaccharides on hematite will bring about significant changes in the electrokinetic behaviour of the minerals, as illustrated in Figs. 4 and 5. Electrokinetic changes on hematite and kaolinite brought about by interaction with mineral-adapted cells and their extracellular products were studied. The following observations are noteworthy: a. Significant negative shifts in hematite IEP could be observed after interaction with hematite- and kaolinite-grown cells. b. IEP of kaolinite shifted to higher pH (2.1 to 3–3.5) after similar interactions.
190
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
15
Hematite before interaction Interacted with EP from hematite-grown cells Interacted with EP from kaolinite-grown cells
30
0
0 0
2
4
6
8
10
-15 -30 -45
a
Zeta potential, mV
15 Zeta potential, mV
Kaolinite before interaction Interacted with EP from hematite-grown cells Interacted with EP from kaolinite-grown cells
0
4
6
8
10
-15
-30
a
-45
pH
2
pH
Fig. 4. Zeta potential of (a) hematite and (b) kaolinite before and after interaction with EP secreted by Bacillus subtilis grown in the presence of minerals as a function of pH. (Note: standard deviation varies between ±0.8 and 2.4 for n = 5.)
30
15
Hematite before interaction
Kaolinite before interaction
Interacted with ECP from hematite-grown cells
Interacted with ECP from hematite-grown cells
Interacted with ECP from kaolinite-grown cells
Interacted with ECP from kaolinite-grown cells
0 0
2
4
6
8
10
-15
-30
a
Zeta potential, mV
Zeta potential, mV
15
0 0
4
6
8
10
-15
-30
-45 pH
2
b pH
Fig. 5. Zeta potential of (a) hematite and (b) kaolinite before and after interaction with ECP secreted by Bacillus subtilis grown in the presence of minerals as a function of pH. (Note: standard deviation varies between ±0.2 and 2.5 for n = 5.)
c. Such IEP shifts observed with hematite and kaolinite are due to bacterial secretion and subsequent adsorption of EP and ECP onto minerals. d. Significant surface adsorption of EP on kaolinite resulted in IEP shift of kaolinite from pH 2.1 to 4.0. Interaction with EP extracted from mineral-grown cell free culture shifted the IEP of hematite from pH 5.5 to about 4.0 e. IEP’s of hematite and kaolinite were shifted to lower pH values with zeta potentials becoming more negative after interaction with ECP extracted from mineral-grown cells. Effect of mineral interaction with extracellular proteins and polysaccharides on the shifts in IEP’s and zeta potentials of hematite and kaolinite was cross checked using BSA and starch as standard proteins and polysaccharide and similar results were obtained. 3.3. Flocculation–dispersion behaviour of hematite and kaolinite Flocculation-dispersion behaviour of hematite and kaolinite particles before and after interaction with EP and ECP extracted from bacterial cells under different growth conditions was then established as shown in Figs. 6 and 7. In the absence of bacterial interactions, settling rate of both hematite and kaolinite decreased sharply with increase in pH. Corresponding to neutral pH conditions, maximum settling (flocculation) for hematite was achieved using ECP extracted from hematite-grown cells
followed by ECP from kaolinite-grown cells. Similarly, maximum dispersion of kaolinite was observed after interaction with EP extracted from either kaolinite- or solution-grown cells at neutral pH. EP extracted from hematite-grown cells did not promote higher kaolinite dispersion. On the other hand, ECP extracted from hematite- and kaolinitegrown cells was also effective in bringing significant dispersion of kaolinite particles. Higher-surface affinity of kaolinite for EP and of ECP for hematite is responsible for the above observed flocculationdispersion behaviour. Flocculation-dispersion of hematite–kaolinite mixtures using different biotreatments was studied in detail and typical results are illustrated in Table 1. Pretreatment with EP extracted from bacterial cells and their metabolites after growth in the presence of hematite brought about significant flocculation of hematite and dispersion of kaolinite. Similar flocculation of hematite as well as kaolinite dispersion could be achieved after interaction with ECP isolated from kaoliniteand hematite-grown cells and their metabolites. Through prior biotreatments with such extracellular polymeric substances, effective selective flocculation of hematite from its mixture with kaolinite could be achieved. The above observations open up newer possibilities in developing a cost-effective, energy-efficient and environment-friendly bioprocess for removal of iron oxides (hematite) from kaolin and china clays. To demonstrate the new bioprocess, iron removal
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
120
120
a
b
Hematite before interaction
Kaolinite before interaction Interacted with EP from
Interacted with EP from
100
Solution-grown cells
Solution-grown cells
Hematite-grown cells
Hematite-grown cells
Kaolinite-grown cells
Kaolinite-grown cells
Percent settled
Percent settled
100
191
80 60 40
80 60 40 20
20
0
0 0
2
4
6
8
0
10
2
4
pH
6
8
10
pH
Fig. 6. Flocculation behaviour of (a) hematite and (b) kaolinite before and after interaction with different bacterial extracellular proteins (EP) as a function of pH. (Note: standard deviation varies between ±1.0 and 4.0 for n = 5.)
140
140 a
Interacted with ECP from
Kaolinite before interaction Interacted with ECP from
120
Solution-grown cells
120
Solution-grown cells
Hematite-grown cells
Hematite-grown cells
Kaolinite-grown cells
100
Percent settled
Percent settled
b
Hematite before interaction
80 60 40
100
Kaolinite-grown cells
80 60 40 20
20
0
0 0
2
4
6
8
0
10
2
4
6
8
10
pH
pH
Fig. 7. Flocculation behaviour of (a) hematite and (b) kaolinite before and after interaction with different bacterial extracellular polysaccharides (ECP) as a function of pH. (Note: standard deviation varies between ±2.0 and 4.0 for n = 5.) Table 1 Flocculation–dispersion of hematite and kaolinite after different biotreatments (note: standard deviation varies between ±1.0 and 3.5 for n=5). Type of biotreatment
Control (no biotreatment) EP from kaolinite-grown cells and metabolites ECP from hematite-grown cells and metabolites ECP from kaolinite-grown cells and metabolites
Percent weight settled in 5 min 1:1 Mixture
Kaolinite only
Hematite only
Kaolinite
Hematite
45 08
60 80
50 10
65 85
05
98
15
95
10
90
20
80
studies were carried out using kaolin clay samples containing up to 2% iron as Fe2 O3 . After 5–10 min interaction with hematite-grown cells of Bacillus subtilis and ECP extracted from the metabolites, up to 80–90% of iron could be removed from the kaolin clay sample through selective flocculation. The developed bioprocess could be compared with existing chemical alternatives such as selective flocculation and flotation using toxic chemicals such as amines and polyacrylamides. Efficient separation of kaolin clays from iron oxides or of iron oxides from clays is not possible through
such methods including high intensity magnetic separation and selective flocculation–dispersion. A bioprocess, on the otherhand, would be more process efficient, cost effective, energy efficient and environment–friendly. 4. Conclusions Bacillus subtilis was grown and adapted in the presence of hematite and kaolinite. Kaolinite-grown (adapted) cells secreted
192
S. Poorni, K.A. Natarajan / Colloids and Surfaces B: Biointerfaces 114 (2014) 186–192
enhanced quantities of mineral-specific proteins and cells grown in the presence of hematite promoted higher secretion of polysaccharides. Extracellular proteins (EP) exhibited higher surface affinity towards kaolinite, which was rendered hydrophobic. Increased adsorption of exopolysaccharides (ECP) onto hematite resulted in enhanced surface hydrophilicity. Significant surface changes in terms of shifts in IEPs and zeta potentials were observed both on bacterial cells and minerals after interaction with EP and ECP. Flocculation of hematite particles in an aqueous medium was found to be promoted after interaction with ECP from mineral-grown cells and their metabolites, while kaolinite was efficiently dispersed after such interactions. Interaction with EP from kaolinite-grown cells also promoted dispersion of kaolinite. Iron oxides such as Fe2 O3 (hematite) which occurs as impurities in kaolin clays can be efficiently removed through prior interaction with EP and ECP from mineral-grown Bacillus subtilis through selective bioflocculation of hematite and dispersion of kaolinite at neutral pH.
Academy of Sciences (India) for Platinum Jubilee Senior Scientist Fellowship to the corresponding author (Prof. K.A. Natarajan). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
Acknowledgements The authors are thankful to the Department of Science and Technology, Government of India, New Delhi for providing financial support to this research work. Thanks are also due to the National
[14] [15] [16] [17] [18]
Y. Cao, X. Wei, P. Cai, Q. Huang, X. Rong, W. Liang, Colloid. Surf. B 83 (2011) 122. E.S. Shelobolina, S.M. Pickering, D.R. Lovely, Clays Clay Miner. 53 (2005) 580. S. Fiore, S. Dumontet, F.J. Huertas, V. Pasquale, Appl. Clay Sci. 53 (2011) 566. L.M.S. de Mesquita, F.F. Lins, M.L. Turem, Int. J. Miner. Process. 71 (2003) 31. I. Styriakova, I. Styriak, Ceramics—Silikaty 44 (2000) 135. C. Cameselle, M.J. Nunez, J.M. Lema, J. Chem. Technol. Biotechnol. 70 (1997) 349. S.K. Mandal, P.C. Banerjee, Int. J. Miner. Process. 74 (2004) 263. M.R. Hosseini, M. Pazouki, M. Ranjbar, M. Habibian, Appl. Clay Sci. 37 (2007) 251. J.E. Kostka, E. Haefele, R. Viehweger, J.W. Stucki, Environ. Sci. Technol. 33 (1999) 3127. K.S. Cho, E.Y. Lee, H.W. Ryu, Appl. Clay Sci. 22 (2002) 47. H.Sarvamangala, K.A. Natarajan, Int. J. Miner. Process. 99 (2011) 70. M.P. Deutscher, Methods in Enzymology Guide to Protein Purification, Academic Press, New York, 1990. D.T. Plummer, An Introduction to Practical Biochemistry, 2, McGraw-Hill, London, 1978. M.M. Bradford, Anal. Biochem. 72 (1976) 248. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350. M.R. Sabari Prakasan, K.A. Natarajan, Colloids Surf., B 78 (2010) 163. D. Fortin, S. Langley, Earth-Sci. Rev. 72 (2005) 1. B.K.G. Theng, Clays Clay Miner. 30 (1982) 1.
