Adhesion of Escherichia coli onto quartz, hematite and corundum: Extended DLVO theory and flotation behavior

Colloids and Surfaces B: Biointerfaces 74 (2009) 140–149

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Adhesion of Escherichia coli onto quartz, hematite and corundum: Extended DLVO theory and flotation behavior Mohsen Farahat a,∗ , Tsuyoshi Hirajima a , Keiko Sasaki a , Katsumi Doi b a b

Department of Earth Resources Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishiku, Fukuoka 819-0395, Japan Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

a r t i c l e

i n f o

Article history: Received 19 February 2009 Received in revised form 9 July 2009 Accepted 14 July 2009 Available online 21 July 2009 Keywords: Adhesion Biosurface modification Extended DLVO theory Flotation Mineral processing

a b s t r a c t The adhesion of Escherichia coli onto quartz, hematite and corundum was experimentally investigated. A strain of E. coli was used that had the genes for expressing protein for silica precipitation. The maximum cell adhesion was observed at pH <4.3 for quartz and at pH 4.5–8.5 for corundum. For hematite, cell adhesion remained low at all pH values. The microbe–mineral adhesion was assessed by the extended DLVO theory approach. The essential parameters for calculation of microbe–mineral interaction energy (Hamaker constants and acid–base components) were experimentally determined. The extended DLVO approach could be used to explain the results of the adhesion experiments. The effect of E. coli on the floatability of three oxide minerals was determined and the results showed that E. coli can act as a selective collector for quartz at acidic pH values, with 90% of the quartz floated at 1.5 × 109 cells/ml. However, only 9% hematite and 30% corundum could be floated under similar conditions. By using E. coli and no reagents, it was possible to separate quartz from a hematite–quartz mixture with Newton’s efficiency of 0.70. Removal of quartz from the corundum mixture was achieved by E. coli with Newton’s efficiency of 0.62. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Utilization of microorganisms as bioreagents in mineral processing (bioflotation or bioflocculation) gave hopes for upgrading minerals from ores by economical and environmentally safe processes. The separation of minerals by biobeneficiation is governed by selective adhesion of microbial cells onto mineral surfaces and the changes occurred on mineral surfaces after the biotreatment. The adhesion of microbial cells onto mineral surfaces is influenced by several properties such as the surface charge, surface free energy components, and the presence and configuration of surface polymers. In general, bacterial adhesion can be explained by surface thermodynamics and the extended DLVO theory, in which the adhesion energy between cells and substrate is calculated as a function of the separation distance [1–3]. These methods take into account Lifshitz–van der Waals interactions, electrostatic interactions, and hydrophobic/hydrophilic force (acid–base) interactions. The most important input in these calculations is the surface energy and its different components (surface charge, Hamaker constants, electron donating, and electron accepting) on the bacterial cell surface and solid substrate. The microbial adherence on mineral surface and the subsequence formation of biofilm result in changes in surface prop-

∗ Corresponding author. Tel.: +81 92 802 7780; fax: +81 92 802 3338. E-mail address: [email protected] (M. Farahat). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.07.009

erties which can be exploited in mineral separation. For example, hematite surface gained hydrophobic properties after being treated with Mycobacterium phlei cells [4,5]. However, hematite gained hydrophilic properties after interaction with Bacillus polymyxa. B. plymyxa rendered quartz hydrophobic properties after sorption on its surface [6]. Escherichia coli was found to be act as a flotation collector for quartz under acidic conditions [7]. In this case, the E. coli strain was genetically modified to express silica-inducing protein (sip), believed to facilitate adhesion and modify mineral surface properties. The sip modified strain of E. coli was derived from a mutation of the E. coli JM109 strain that has sip cloned in the pET32 vector. The sip genes were cloned from the hyperthermophilic bacteria Thermus thermophilus [8]. Hematite and corundum are important industrial minerals; hematite is the principle source of iron. Huge amounts are mined annually for industrial production. Corundum is used as an abrasive in the manufacture of sandpaper, polishing components and cutting tools. The presence of silica in association with hematite and corundum as gangue mineral, affects on their commercial uses. From this point of view, this study investigates the adhesion behavior of E. coli strain sip onto the three oxide minerals experimentally and theoretically by extended DLVO theory. Surface energy components of the microbe and the mineral samples (Hamaker constants and acid–base components) were experimentally determined from contact angle measurements. Subsequent changes on the min-

M. Farahat et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 140–149

eral surface after biotreatment were followed by measuring the mineral zeta potential and contact angles. Flotation behavior and possibility to remove silica from hematite/corundum mixture were investigated through single mineral and differential microflotation experiments. 2. Materials and methods 2.1. Mineral samples Hand-picked natural pure samples of quartz, hematite, and corundum were obtained from different locations. Quartz was obtained from Ishikawa Prefecture, Japan, while the hematite sample was obtained from the Bahariya Oasis, Egypt. The ruby corundum sample was from Southern District, Madagascar. The samples were crushed with a hammer and ground in a planetary ball mill (Fritch pulverisette 6). The ground samples were sieved to obtain the −105 ␮m + 78 ␮m size fraction for flotation experiments and the −38 ␮m fraction was further classified by sedimentation to remove particles of diameter less than 5 ␮m. The −38 ␮m + 5 ␮m fraction was used for adsorption and electrokinetic experiments. 2.2. Bacterial strain A pure culture of E. coli strain sip was used in this study. The strain was cultured in lysogeny broth (LB) medium consisting of tryptone (10.0 g/l), yeast extract (5.0 g/l), and sodium chloride (10.0 g/l). Cultures were grown in shake flasks at 150 rpm and at 37 ◦ C. The cell concentration was determined by measuring the optical density at 660 nm on a Taitech Miniphoto 518R spectrophotometer. When the OD660 reached 0.6, 2 mM of IPTG was added, and growth was continued for a further 3 h. Cells were harvested by centrifugation (Tomy SRX-201) at 6680 × g for 20 min. The cell pellets were washed three times with 2 mM Tris buffer and then stored in a refrigerator at 4 ◦ C until used in adsorption and flotation experiments. 2.3. Electrokinetic measurements After conditioning at the required pH values, the zeta potential of quartz, hematite, and corundum samples and bacterial cells was measured using the ZEECOM ZC-2000 system (Kyowa Interface Science Co., Ltd.) equipped with a video recorder. The zeta potential of bacterial cells was measured at a concentration of 108 cells/ml. All measurements were conducted at the same ionic strength (10−3 M KNO3 ). To measure the zeta potential of mineral samples after interaction with bacteria, the sample was first conditioned with bacteria under the required conditions (pH, adsorption time, and cell concentration), and unattached cells were removed by centrifugation at 400 × g for 3 min. The residual mineral particles were resuspended in 10−3 M KNO3 , and the pH was readjusted to the initial value, after that, the suspension was allowed to stand for ten minutes until the coarse particles settle down, and the remained fine particles were used for zeta potential measurements. 2.4. Adsorption experiments The microbial cells were adsorbed onto quartz, hematite, and corundum in 100-ml Erlenmeyer flasks containing 0.5 g of the mineral (−38 ␮m + 5 ␮m fraction) in 50 ml of 10−3 M KNO3 . Cells were added to the suspension to achieve the initial target cell concentration at the required pH. The slurry was conditioned by mechanical shaking at 120 rpm and at 25 ◦ C for different time intervals of 3–30 min. Unattached cells were counted microscopically using a Petroff–Hausser counter, and the number of adsorbed cells was calculated from the difference between the initial and final cell numbers.

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2.5. Contact angle measurements The contact angles were measured using a goniometer (Dropmaster 300, Kyowa Interface Science Co., Ltd.). Samples were prepared using the method reported by Misra et al. [9]. The surface of each mineral sample was carefully polished using distilled water and alumina powder. The polished samples were cleaned with jets of distilled water to remove any particles sticking to the surface. The contact angles of the mineral samples were measured before and after treatment with the microbe. To measure the contact angles after biotreatment, cell suspensions were freshly prepared in 10−3 M KNO3 and the pH was adjusted to 2.5 using HNO3 . The cell suspension was conditioned together with the mineral sample on a rotary shaker for 30 min. The solids were separated from the mixture and dried at room temperature. For measuring the contact angle of bacteria, bacterial lawn was prepared by filtering bacterial suspension on a filter membrane, about 800 bacterial layers were depositing on the filter membrane. The filter membrane with the bacterial lawns was cut into strips and fixed with double-sided adhesive tape onto a sample holder. Contact angles were measured using the sessile drop method. A 2-␮l drop of a liquid was placed on the surface, and measurements were recorded. The average value of six readings is reported here. 2.6. Flotation experiments Microflotation tests were carried out in a modified Hallimond tube [10]. The tests were divided into two categories: flotation with single minerals and flotation with 1:1 mixtures of quartz–hematite and quartz–corundum. In each experiment, 1 g of mineral (in the single mineral flotation experiments) or 0.5 g of each mineral (in the binary system experiments) was conditioned with a known bacterial cell concentration in 130 ml 10−3 M KNO3 at a specific pH value for 10 min. The size fraction of the mineral particles in these experiments was −105 ␮m + 78 ␮m. The solution was transferred into a Hallimond tube, and flotation was initiated by passing N2 at a flow rate of 20 ml/min. The floated and tailing fractions were collected separately, dried, and chemically analyzed. 3. Results and discussion 3.1. Estimation of interaction energies by extended DLVO theory The interaction energy between the microbe and quartz, corundum, and hematite was calculated using the extended DLVO theory. The classical DLVO theory as described by Verwey and Overbeek [11] and Deryagin and Landau [12] includes attractive or repulsive electrostatic forces and Lifshitz–van der Waals attractive forces (LW). The acid–base interaction component, which is based on electron donating and electron accepting interactions between polar moieties in aqueous solution, was added by van Oss et al. [13] to formulate the extended DLVO theory (XDLVO). According to the extended DLVO theory, the total interaction energy (GTot ) is determined as GTot = GEL + GAB + GLW

(1)

where GEL is the repulsion potential due to the formation of an electrical double layer around particles and cells, and GAB is the acid–base interaction energy and GLW is the Lifshitz–van der Waals attractive energy. 3.1.1. Electrostatic interaction energy GEL The electrostatic repulsive force GEL between microbial cell and mineral particle can be expressed by the following equation for the

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M. Farahat et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 140–149 Table 1 Contact angle data for minerals and bacterial cells.

sphere–sphere system:



 2

εa1 a2 12 + 2

GEL =





1 + exp(−H) × ln + ln{1 − exp(−2H)} 1 − exp(−H) 12 + 22 21 2

(2)

where ε is the dielectric constant of the aqueous medium a1 and a2 represent the radius of the cell and mineral particle, respectively;  1 and  2 are the surface charges of the mineral and cell, respectively; H is the minimum separation distance between the cell and particle; and −1 is the double layer thickness that can be calculated from the following equation:

 =



e2 εKT

1/2

where ε is the tron charge, K temperature, zi number of ions reduced to







(3)

permittivity of the medium e denotes the elecis the Boltzmann constant, T is the absolute is the valency of the ions present, and n is the per unit volume.Assuming a2  a1 , Eq. (2) can be

GEL = εa1 12 + 22 ×



m−1

izi ni

21 2 12 + 22



ln



1 + exp(−H) + ln{1 − exp(−2H)} 1 − exp(−H)

(4)

3.1.2. Acid–base interaction energy GAB The acid–base interaction energy GAB for the particle–cell system can be calculated according to the following equation (sphere–sphere system): AB GAB = a(Gadh )e[(d0 −H)/]

(5)

where a is the radius of the solid particle,  is the correlation length of the molecules in the liquid (∼6 Å), d0 is the minimum separation distance between two surfaces (1.57 Å), and H is the separation distance. AB can be calculated as follows from either the geometric Gadh mean approach AB Gadh = −2



bAB −



AB w



AB − m



AB w

 (6)

AB Gadh =2

−2 −2

b+ −







b+ − + m −

+ m



b− −







 

+ w ( + w



b− −

− m



− m −



− w

Water

Formamide

1-Bromonaphthalene

45.0 35.0 41.8 53.0

36.5 29.6 32.4 35.8

18.4 21.0 22.1 36.2

To calculate these parameters ( + ,  − and Lifshitz–van der Waals surface tension component  LW ) for the cell or minerals, the contact angles of three different liquids (water, formamide, and 1bromonaphthalene) on the solid phase (mineral or bacterial lawn) were measured, and the parameters were calculated by applying Young’s equation [14]: 1 (1 + cos )l = 2





sLW lLW +

s+ l− +



s− l+

(8)

where s and l refer to solid (mineral or bacteria) and liquid, respectively. Table 1 lists the measured contact angles and Table 2 lists the calculated energy parameters. From the results shown in Table 2, it can be seen that the  + electron acceptor values of the minerals and microbe are less than their  − values. However, there is a significant difference in the  − electron donor value. Previously it has been suggested [14] that the  + values have no role in determining the hydrophilicity or hydrophobicity of the substratum. Instead, the value of the electron donor component  − determines the nature of the surface. If the  − value of a substance is ≥28.3 mJ/m2 , the substance is hydrophilic, and if it is <28.3 mJ/m2 , the substance is hydrophobic. The results indicate that the microbial cells have hydrophobic properties, while minerals are hydrophilic. This conclusion also can be observed from contact angle results in Table 1. 3.1.3. Lifshitz–van der Waals interaction energy For a sphere–sphere interaction, the Lifshitz–van der Waals attractive energy can be obtained from the following equation: GLW =

−a1 a2 Abwm 6H (a1 + a2 )

(9)

Considering that the radius of the mineral particle is larger than that of the bacterial cell, Eq. (9) can be reduced to −a1 Abwm 6H

(10)

where a1 and a2 represent the radius of the cell and mineral particle, respectively, and Abwm is the effective Hamaker constant for the bacteria–water–mineral system.

− w )



Hematite Quartz Corundum Microbe

GLW =

or the Lifshitz–van der Waals acid–base approach



Contact angles (◦ )

Mineral

(a1 + a2 )

 (7)

In these equations,  + and  − refer to electron acceptor and electron donor parameters, respectively, and b, m, and w represent bacteria, mineral, and water, respectively.

3.1.4. Calculation of Hamaker constants There are two methods to calculate the effective Hamaker constants, 3.1.4.1. Method (1) from the individual Hamaker constants. The individual Hamaker constants of the three minerals are known from

Table 2 Calculated energy components of mineral samples and cells (mJ/m2 ). Mineral

 (surface energy)

 LW (van der Waals)

 + (electron acceptor)

 − (electron donor)

 AB (acid–base)

Hematite Quartz Corundum Microbe

47.5 50.0 52.1 47.2

42.1 41.5 42.2 36.2

0.2 0.4 0.7 1.3

34.5 42.4 33.2 23.5

5.4 8.5 9.9 10.9

M. Farahat et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 140–149 Table 3 The individual and effective Hamaker constants for different systems. Material

A (J)

Abwm (J), method (1)

Abwm (J), method (2)

0.4 × 10−20 1.1 × 10−20 0.7 × 10−20

0.4 × 10−20 0.5 × 10−20 0.5 × 10−20

−20

3.7 × 10 8.8 × 10−20 25.0 × 10−20 15.2 × 10−20 5.2 × 10−20

Water Quartz Hematite Corundum Microbe

the literature [15,16], and the Hamaker constant of a microbial cells can be calculated, Fowkes [17] proposed the following equation: Abb = 6r 2  LW

(11) 6r2

where r is intermolecular distance. The value of can be taken as 1.44 × 10−18 m2 and  LW is the Lifshitz–van der Waals energy component. The effective Hamaker constant Abwm can be calculated as follows: Abwm = Abm + Aww − Awm − Awb

(12)

where b, w, and m represent bacteria, water, and mineral, respectively, and Abm = Awm = Awb =



Abb −

 

2



Amm

Aww −

Aww −



(13)

2 Amm

(14)

2



Abb

(15)

Eq. (12) can be written as follows: Abwm =



Abb −



Aww

  ×

Amm −



 (16)

Aww

3.1.4.2. Method (2) from Lifshitz–van der Waals free energy. According to this method, the effective Hamaker constant Abwm is given by the following equation: LW Abwm = −12d2 Gadh

(17) LW Gadh

where d is the minimum separation distance and is the Lifshitz free energy of adhesion which can be evaluated by the following equation: LW = −2 Gadh



bLW −



LW w



LW − m



LW w

 (18)

surface is positively charged in the acidic and alkaline regions, and the IEP is around pH 8.0. These results are in agreement with previously reported data [19]. The zeta potential curve of hematite is similar to that of the cells, and its IEP is around pH 4.0. The IEP for quartz particles could not be determined under these experimental conditions; however, the IEP for quartz has been reported to be approximately pH 2.0 [20]. The total interaction energies between the microbial cells and mineral particles were calculated at different pH values as a function of the separation distance between the cell and particle in 10−3 M KNO3 . The typical results are shown in Figs. 2–5. At all pH values, the primary contribution to the total energy comes from the electrostatic energy GEL , followed by the acid–base energy GAB and the Lifshitz–van der Waals energy GLW . The acid–base interactions are relatively short-range, and the interacting surfaces must be close (less than 5 nm) before these forces become operative. GLW was almost the same at any given pH. For quartz, GEL is negative at acidic pH values, i.e., when the pH <4.3, and GLW is also negative in the same pH range. The summation of these two components and the acid–base energy gives GTot with a net value, indicating that the adhesion between cells and quartz particles in this pH range is thermodynamically preferred. There is a secondary minimum at pH 4.5, which means that the cells can approach to the mineral surface but without adhesion because of the high potential value (reversible adhesion). At pH values higher than 4.5, GEL has positive values and the total interaction energy GTot is positive, indicating that the repulsive forces dominate and the number of adsorbed cells decreases. In the hematite–cell system, GEL is always positive except around pH 4.0, which is close to the IEP of the cell and mineral. GLW has negative values, but because of the high magnitude of GAB , the GTot was always positive at any given pH and the lowest GTot was near the IEP, indicating that the repulsive forces are dominant and hinder the adsorption process. For the corundum–cell system, GTot is positive in two pH regions, i.e., 2.0–4.5 and 8.5–12.0 and is negative in the range 4.5–8.5. This means that adsorption is favored only in the pH range 4.5–8.5. Fig. 6 shows the effect of pH on the total interaction energy of the mineral–microbial cells adhesion at a separation distance of 5 Å. It is clear that selective adsorption between microbial cells and quartz can be achieved in the pH range 2.0–4.0. The corresponding pH range for corundum is 4.5–8.0, while no adsorption can be achieved for hematite. On the basis of these results and in the absence of other interactions, selective adsorption of the microbial cells on the three oxide minerals is expected.

where  LW is the Lifshitz–van der Waals energy component and b, m, w stand for bacteria, mineral and water, respectively. The effective Hamaker constants evaluated by methods (1) and (2) are summarized in Table 3. In this study, Hamaker constants evaluated by method (2) were used. 3.2. Zeta potential measurements before adsorption and interaction energy Fig. 1 shows the zeta potential of microbial cells, quartz, hematite, and corundum as a function of the pH. The results represent averages of three measurements and the standard deviation was ±3 mV. E. coli is a Gram-negative bacterium that has a welldefined cell wall composed of components such as peptidoglycan, lipopolysaccharides, lipoproteins, enzymes, and mycolic acid [18]. The presence of these biomolecules imparts charges (that differ in magnitude and sign) to the bacterial surface depending on the pH. The microbe has positive charges in the acidic region and negative charges in the alkaline region; its iso electric point (IEP) is at pH 4.5. The zeta potential curve for corundum shows that the mineral

143

Fig. 1. Zeta potential curves of minerals and cells as a function of pH.

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onto quartz, corundum, and hematite was 16.0 × 109 , 8.9 × 109 , and 5.2 × 109 cells/m2 , respectively. The effect of pH on the number of cells adsorbed onto quartz, hematite, and corundum minerals is shown in Fig. 8. These experiments were carried out at an initial cell concentration of 2.2 × 108 cells/ml in the presence of 10−3 M KNO3 and a conditioning time of 10 min. In the case of quartz, the number of adsorbed cells was high in acidic regions (lower than pH 4.3) and decreased with an increase in the pH. This can be explained as follows: In the acidic pH region, the total interaction energy GTot is negative as shown in Fig. 6, indicating that the adhesion between cells and quartz particles in this pH range is thermodynamically preferred. At pH values >4.3, GTot is positive, indicating that the repulsive forces dominate and the number of adsorbed cells decreases.

Fig. 2. Variations in the components of the interaction energy of oxide mineral–microbe as a function of the separation distance at pH 2.5.

3.3. After adsorption The effects of the conditioning time and pH on the number of cells adsorbed onto quartz, hematite, and corundum were examined. The experiments were run in duplicate the results represent the average with standard deviation of ±0.4 × 108 cells/ml. Fig. 7 shows the number of cells adsorbed/m2 of the mineral as a function of the interaction time. These experiments were carried out at pH 5.2 in the presence of 10−3 M KNO3 . The initial cell concentration was 2.2 × 108 cells/ml. The number of adsorbed cells increased with time for up to 10 min before saturation in the case of both minerals. Further, the number of cells adsorbed onto quartz was higher than that on corundum and hematite. After conditioning for 5 min, the number of cells adsorbed onto quartz, corundum, and hematite was 14.0 × 109 , 8.0 × 109 , and 3.3 × 109 cells/m2 , respectively. After conditioning for 10 min, the number of cells adsorbed

Fig. 3. Variation in the components of the interaction energy for oxide mineral–microbe as a function of the separation distance at pH 4.5.

M. Farahat et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 140–149

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porous surface); second, the cells are adsorbed onto the silica traces which are found in hematite sample as impurities; third, the cells are adsorbed at the secondary minimum as mentioned previously. The conclusions obtained from the interaction energy using the data based on before adsorption, approximately explain the results of the adsorption experiments of quartz, hematite, and corundum. Fig. 9 shows the zeta potential curves of the three minerals after interaction with the microbe. The initial cell concentration was 2.2 × 108 cells/ml. For quartz, significant changes occur after interaction with the cells, and the IEP of quartz increases from less than 2.0 to approximately 4.5. Moreover, the surface charge of quartz particles becomes highly positive under acidic conditions. The zeta potential of quartz after conditioning with bacteria is close to that of the bacteria, especially under acidic conditions. This indicates that adsorption of cells onto the quartz surface may influence

Fig. 4. Variations in the components of the interaction energy for oxide mineral–microbe as a function of the separation distance at pH 8.5.

In the case of corundum, the number of adsorbed cells was lower than that on quartz at pH <5.0. In the pH range 5.5–9.0, the number of adsorbed cells was slightly higher than that on quartz and hematite. At a pH >9.0, the number of adsorbed cells was very low, i.e., there was almost no adsorption. For the corundum–cell system, GTot is positive in two pH regions, i.e., 2.0–4.5 and 8.5–12 and is negative in the range 4.5–8.5 as shown in Fig. 6. This means that adsorption is favored only in the pH range 4.5–8.5. At low pH, there is a possibility of a “secondary minimum” where a much weaker and potentially reversible adhesion between corundum and cells exists (Fig. 3). In the case of hematite, the number of adsorbed cells was low at all pH values except for the region close to IEP of the mineral. The increase in the cell number in adsorption experiments can be attributed to three reasons; first, the immobilization of the cells into hematite porous (SEM images showed that hematite has a

Fig. 5. Variations in the components of the interaction energy for oxide mineral–microbe as a function of the separation distance at pH 10.5.

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Fig. 6. Effect of the pH on the total interaction energy of mineral–microbe adhesion at a separation distance of 5 Å. Fig. 9. Zeta potential curves of mineral samples after treatment with cells as a function of the pH (cell concentration was 2.2 × 108 cells/ml, 10 min.).

Fig. 7. Adsorption of cells onto oxide minerals as a function of the conditioning time (cell concentration 2.2 × 108 cells/ml, pH 5.2).

It is evident that for 107 cells/ml the average zeta potential of quartz changed slightly from −25 to −22 mV. However, for concentrations of 5.0 × 107 and 108 cells/ml, quartz showed a mixture of positively and negatively charged particles, with an average zeta potential of −1.0 and +7.0 mV, respectively. A further increase in bacterial concentration led to one charge phase (with all particles positively charged) and the average zeta potential changed to +18.0 mV for 2.2 × 108 cells/ml and +21.0 mV for 5.0 × 108 cells/ml. The variation in zeta potential of quartz particles with bacterial concentration is likely due to changes in surface coverage. For hematite, almost no significant changes occurred in the zeta potential of hematite after interaction with the cells. There are two possible explanations for this. First, microbial cells and hematite particles have similar surface potentials, indicating that even if there is adsorption, changes in their surface potential would not be noted. Second, due to the similarity in the surface potentials, the generated repulsion forces hinder the adsorption of bacterial cells to the mineral surface, and the number of adsorbed cells is insufficient to change the surface potential of hematite. For corundum, significant changes are observed in the pH range 4.0–8.0. The IEP of corundum shifts from pH 8.2 to pH 5.0, and the zeta potential of corundum is reversed after interaction with the cells. This indicates that at this pH range, the number of adsorbed cells is sufficient to cause changes in the surface potentials of

Fig. 8. Adsorption of cells onto quartz, hematite and corundum as a function of the pH (cell concentration was 2.2 × 108 cells/ml, 10 min.).

the surface properties of quartz surface; making it more similar to those of the bacterial surface. This may occur through the formation of bacterial layer on the quartz surface. The relationship between the initial cell concentration and the zeta potential distribution of quartz particles is shown in Fig. 10. These experiments were carried out at pH 2.5 in 10−3 M KNO3 at different cell concentrations.

Fig. 10. Distribution of quartz zeta potentials after interaction with bacteria as a function of cell concentration under pH 2.5, 10−3 M KNO3 .

M. Farahat et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 140–149

Fig. 11. Distribution of corundum zeta potentials after interaction with bacteria as a function of cell concentration under pH 5.7, 10−3 M KNO3 .

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Fig. 12. Floatability of biotreated minerals as a function of the pH (cell concentration 2.2 × 108 cells/ml).

3.5. Flotation experiments corundum. Fig. 11 shows the relationship between the initial cell concentration and the zeta potential distribution of corundum particles, These experiments were carried out at pH 5.7 in 10−3 M KNO3 at different cell concentrations. For 107 cells/ml the average zeta potential of corundum changed from +20.0 to +16.0 mV. However, for concentrations of 5.0 × 107 and 108 cells/ml, corundum showed a mixture of positively and negatively charged particles, with an average zeta potential of +12.6 and 0.0 mV, respectively. A further increase in bacterial concentration led to dramatic change in the zeta potential of corundum where the average zeta potential changed to −7.8 mV for 2.2 × 108 cells/ml and −9.0 mV for 5.0 × 108 cells/ml. 3.4. Contact angle measurements Table 4 summarizes the contact angles for quartz, corundum, and hematite before and after interaction with the cells (5.0 × 109 cells/ml) at pH 2.5. The results represent average of six readings and the standard error was ±2◦ . The surface of quartz is hydrophilic prior to interaction with the cells, and its contact angle is 35◦ . However, after interacting with the cells, this material gains hydrophobic characteristics, and the contact angle shifts by more than 20◦ . This is because the number of cells adsorbed onto the surface of quartz is sufficient to impart their own hydrophobic surface properties to quartz through the adhesion of bacteria. Small changes are observed in the contact angles of hematite and corundum after interaction with the cells, and the contact angle shifts from the initial value by approximately 5–7◦ in the case of both hematite and corundum. The number of cells adsorbed onto hematite and corundum at this pH is not sufficient to cause significant changes in their surface properties. The results of the contact angle are compatible with those of extended DLVO theory as shown in Fig. 6 and those of adsorption experiments as shown in Fig. 8.

Table 4 The contact angles of quartz, hematite, and corundum before and after interaction with sip E. coli at pH 2.5. Mineral

Quartz Hematite Corundum

Contact angles (◦ ) Untreated

Biotreated

35.0 45.0 41.8

58.0 49.7 48.0

3.5.1. Single mineral flotation Fig. 12 shows the results of biotreated single mineral flotation tests. Experiments were carried out at a cell concentration of 5.0 × 108 cells/ml in the presence of 10−3 M KNO3 for a conditioning time of 10 min at different pH values and a flotation time of 3 min. The experiments were run in duplicate and the results represent the average with standard deviation of ±2.5%. The results showed that it is possible to float biotreated quartz at pH <4.3 with a recovery of 58% under these experimental conditions. These results are compatible with the extended DLVO theory calculations shown in Fig. 6. The results also are in agreement with those of the adsorption experiments in which large numbers of cells adsorb onto the quartz surface at low pH values, and the quartz particles gained hydrophobic characteristics. Consequently, the flotation recovery was high. The flotation recovery of biotreated corundum was lower than that of quartz. Maximum recovery was obtained in the pH range 5.0–8.0, which is the same range in which the total interaction energy is a negative and high adsorption density is achieved. Biotreated hematite did not float at any pH value, and its flotation recovery was less than 10%. The adsorption of cells onto the mineral surface influences the surface properties of mineral surface; making it more similar to those of the bacterial surface. Because the E. coli strain sip surface is a hydrophobic, the mineral surface changes to more hydrophobic surface. In low pH, there is a possibility of a “secondary minimum” where a much weaker and potentially reversible adhesion between cells and particles exists (Fig. 3). These weak adhesions are sufficiently stable not to be detached by Brownian motion, but may dissociate under an externally applied force such as vigorous agitation. From these considerations, it was expected that the flotation results are similar to the results estimated from Fig. 6. In the acidic pH region, the total interaction energy GTot of quartz is negative, indicating that the adhesion between cells and quartz particles in this pH range is thermodynamically preferred and the quartz surface changed to hydrophobic. For the corundum–cell system, GTot is negative in the range 4.5–8.5 and for hematite, GTot is positive in all the pH range. This means that flotation is favored only in the pH range 4.5–8.5 for corundum, and in the acidic pH range for quartz. Fig. 13 shows the effect of the bacterial concentration on the flotation recovery of biotreated minerals (single mineral tests). The experiments were carried out at pH 2.5 in the presence of 10−3 M KNO3 . The flotation recovery of quartz increased with an increase in the cell concentration, and 40% of the quartz could be recovered with 5 × 107 cells/ml. The recovery increased proportionally with

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Fig. 13. Floatability of biominerals as a function of the cell concentration at pH 2.5.

an increase in the cell number and reached its maximum value at 1.5 × 109 cells/ml, where 90% of quartz was recovered in the float fraction. The increase in cell concentration had a slight effect on the flotation recovery of corundum. The maximum recovery was 35%, and this was obtained with 1.5 × 109 cells. The flotation recovery of hematite was unaffected by increasing the cell number. At a cell density of 1.5 × 109 cells/ml, only 9% of the hematite sample was recovered in the float fraction. This can be explained on the basis of the adsorption data shown in Fig. 8 and contact angle data in Table 4. It is clear that the number of cells adsorbed onto quartz is much higher than that adsorbed onto hematite or corundum, and the contact angle of biotreated quartz is higher than that of both hematite and corundum. 3.5.2. Differential flotation experiments 3.5.2.1. Hematite–quartz system. Fig. 14 shows the effect of the bacterial concentration on the grade and recovery of hematite and quartz in the tailing fraction. As the bacterial concentration increases, the quartz floats and the quartz grade in tailing fraction decreases from 52% to 17%. However, in the case of hematite, since quartz is removed in the float fraction, the grade of hematite in the tailing increases from 48% to 80% with 88% recovery. The highest quartz grade in the float fraction (92% with 76% recovery) is achieved at 5.0 × 108 cells/ml. When the cell concentration increases to 109 cells/ml, the grade in the float fraction decreases to 85%, and the recovery increases to 82%.

Fig. 14. Distribution of quartz and hematite in the tailing fraction as a function of the cell concentration at pH 2.5.

Fig. 15. Distribution of quartz and corundum in the tailing fraction as a function of the cell concentration at pH 2.5.

3.5.2.2. Corundum–quartz system. Fig. 15 shows the effect of the bacterial concentration on the grade and recovery of corundum and quartz in the tailing fraction. As the bacterial concentration increases, the quartz floats and the grade decreases. The grade decreases from 49% at no cells to 22% at 109 cells/ml. However, in the case of corundum, the removal of quartz from the tailing fraction results in an increase in the grade of corundum in the tail from 52% to 80% with 87% recovery. The highest grade of quartz in the float fraction (84% with 72% recovery) is achieved at 109 cells/ml. To assess the separation efficiency in the differential flotation experiments, Newton’s efficiency was calculated. This can be derived from the following equation:

= a + b − 1

(19)

where is Newton’s efficiency,  a is the recovery of hematite/corundum in the tailing, and  b is the recovery of quartz in the float. The results are shown in Fig. 16. It is clear that when bacteria are not used, Newton’s efficiency is almost 0 in both systems, indicating no separation. However, with increasing cell concentration, Newton’s efficiency increases proportionally and reaches its maximum value of 0.70 at a cell concentration of 5.0 × 108 cells/ml for the hematite–quartz system. The maximum Newton’s efficiency is 0.62 for the corundum–quartz system at a cell concentration of 109 cells/ml.

Fig. 16. Effect of the bacterial concentration on Newton’s efficiency.

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4. Conclusions

Acknowledgments

The results of the adhesion experiment show that E. coli strain sip has higher affinity to quartz than to hematite or corundum, especially at pH <4.3. However, in the alkaline region, the number of cells adsorbed onto quartz and other minerals decreases. The calculated energy components of mineral samples and cells show that the microbial cells have hydrophobic properties, while the minerals are hydrophilic. The effect of pH on the total interaction energy of the mineral–microbial cells adhesion was calculated based on the zeta potential measurements before adhesion. The extended DLVO approach predicts adhesion of the microbe on quartz mineral at pH <4.3 and on corundum at pH 4.5–8.5, but no adsorption on hematite regardless the pH. These phenomena were observed in the adsorption and flotation experiments, as the maximum adsorption on quartz was at pH <4.3, and for corundum was at pH 4.5–8.5 and low adsorption for hematite. Surface changes occurred on the mineral surface after treatment with the cells. For quartz, significant changes were obtained, the IEP shifted from less than 2.0 to 4.3. Moreover, the contact angle changed from 35◦ to about 58◦ . No significant changes were noticed in the case of hematite. There were significant changes in the zeta potential of corundum when its IEP shifted from 8.2 to 5.0. Single mineral flotation experiments showed that the maximum flotation recovery for quartz was obtained at pH <4.3, and for corundum, the maximum recovery was at pH 4.5–8.5 range. However, for hematite, the recovery was very low over all pH. By using E. coli strain sip and no other reagents, it was possible to separate quartz from a hematite–quartz mixture with a Newton’s efficiency of 0.70, and the grade of hematite increased from 48% to 80%. Removal of quartz from the corundum mixture was also possible. After biotreatment, the grade of corundum increased from 52% to 80% with a Newton’s efficiency of 0.62.

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