Bio‐flotation of Egyptian Phosphate Using Desulfvibrio Desulfuricans Bacteria

Journal of Mining World Express (MWE) Volume 4, 2015 www.mwe‐journal.org doi: 10.14355/mwe.2015.04.002

Bio‐flotation of Egyptian Phosphate Using Desulfvibrio Desulfuricans Bacteria N. A., Abdel‐Khalek, K. A. Selim, K. E. Yassin, S. S. Abdallah Central Metallurgical Research & Development Institute, Helwan, Cairo, Egypt, P.O. Box.87 Helwan – Cairo‐Egypt naguialy@gmail.com; k2selem1@gmail.com; khaled_yassin@yahoo.com; samahsaleh86@gmail.com Abstract The bio‐flotation and bio‐flocculation processes concern the mineral response to the bacterium presence, which is essentially considered as interplay between microorganism and the physicochemical properties of the mineral surface. The adhesion of microorganisms to minerals results in alteration of surface chemistry of minerals relevant to beneficiation process due to a consequence of the formation of a biofilm on the surface or bio‐catalyzed surface oxidation or reduction products. In this paper, the amenability of utilization of Desulfvibrio desulfuricans isolated and adapted on surfaces of phosphate ore, as flotation reagents for separating silica from apatite in the bio‐flotation of quartz‐apatite minerals system has been studied. The effect of this bacterial isolates on the surface properties of the two single minerals has been studied through zeta potential and adhesion measurements as well as micro‐flotation tests. The effect of pH of the medium on the surface properties and flotation behaviour of each single mineral is determined. Flotation of binary mixtures of quartz‐apatite as well as natural phosphate ore has also been performed at different operating parameters. Different characterization techniques for both single minerals and bacteria isolated from their surfaces have been done using XRD, SEM, and contact angle as well as morphological and biochemical identification of bacterial isolates.

Keywords Bio‐Flotation; Phosphate Ore; Apatite; Quartz; Desulfovibrio Desulfuricans Bacteria and Surface Modification

Introduction Phosphate rocks are vital nonrenewable resources and are essential components in agricultural fertilizers and phosphorous‐based chemicals. About 85% of the phosphate produced is consumed in the fertilizer industry.

Phosphate deposits can be divided into three groups: sedimentary, igneous and biogenetic deposits [1]. In the recent years, mining industry has been facing several problems that influence the mineral processing, such as the depletion of high‐grade ores [2] and environmental regulations [3]. The former aspect compels the mining industry to process low grade ores, fine mineral particles and flotation tailings to produce material suitable for a global market. Thus, it has become very important to develop appropriate and environmentally friendly technologies able to complement the conventional techniques used at the mineral concentration. In this context, bio‐beneficiation is increasing its role in mineral processing. The main purpose of this procedure is to selectively undertake the removal of undesirable mineral constituents from an ore, through interaction with microorganisms and/or their metabolic products, thus enriching it, with respect to the desired valuable minerals [4‐6].

Bio‐flotation is becoming very attractive for presenting a great technological potential, environmental acceptability, flexibility in the choice of microorganisms and especially due to its mineral selectivity [2, 5] and also for processing fine and ultra‐fine mineral particles. One of the most important steps in mineral bio‐flotation is the adhesion of the bacteria onto the mineral surface [5, 7]. The bacterial adhesion occurs as a net result of attractive and repulsive forces of the cell and mineral surfaces. The interactions that result in such adhesion include electrostatic interactions, acid–base interactions, van der Waals forces and hydrophobic interactions, all of which are determined by the cell‐wall and mineral surface properties. The surface modification can be direct or indirect; the direct mechanism involves the adhesion of the bacterial cells to mineral particles, while the indirect mechanism refers to metabolic products which act like surface activator reagents. Both interactions may allow the mineral surface to acquire hydrophobic or hydrophilic properties [8, 9]. This paper aims at studying the role of bacterial isolate of Desulfvibrio desufuricans in bio‐flotation of apatite‐quartz system in relation to Egyptian phosphate ore.

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The role of bacteria on the surface properties of the two single minerals has been studied through zeta potential, adsorption, surface tension and adhesion measurements as well as micro‐flotation tests. Flotation of binary mixtures of apatite‐quartz as well as Egyptian natural phosphate ore has also been performed.

Materials and Methods Materials Samples of single minerals of quartz (SiO2), apatite Ca5 (PO4)3(OH,F,Cl) were delivered from ‘Wards’ Company, USA. The purity (99.9 %) of the samples was confirmed using XRF. The –200 mesh fractions were used in adsorption and flotation studies. Analytical grade HCl and NaOH, from Aldrich, were used for pH regulations.

Natural phosphate ore was collected from New Valley, Egypt.

Methods 1)

Microorganism Growing and Isolation

28 gm of bacterial medium consisting of peptone, beef‐extracted, NaCl and mycological Agar was dissolved in 1L of bi‐distilled water. The solution was autoclaved at 120oC, cooled and poured in Petri dish for solidification.

After that, about 0.5 gm from the autoclaved solid was suspended in 100 ml of bi‐distilled water; 1 ml of solution was poured on agar plate and then incubated at a temperature of 37°C for 24 – 48 hours. Appearing different colored spots indicated the presence of different types of microorganisms. The microorganisms were grown in a bacterial medium without agar and incubated for 24 hr at 37°C. The bacterial population can be determined by measuring the turbidity or the optical density of the bacterial suspension using a UV visible spectrophotometer (Lambda 3B, Perkin‐Elmer). Because the turbidity is directly proportional to the number of cells, this property was used as an indicator for bacterial concentration.

The cells suspended in the suspension to interrupt the passage of light and allow less light to reach the photoelectric cell and the amount of light transmitted through the suspension was measured as percentage transmission. The turbidity for cell suspension is measured at a wavelength of 550 μm against clear water as reference, at which the 0.01 reading is equivalent to 106 cells/ mL. Six micro‐liters of bacteria was centrifuged at 16000 rev. /min. and rinsed three times with double distilled water and thereafter, diluted in 100 ml double distilled water [8, 10 and 11]. Different types of bacteria were isolated, grown, counted and their efficiency was screened using a laser particle size analyzer. Based on the later test, one of these types of bacteria (which showed the best efficiency) was selected to conduct this study. Microbiological investigations indicated that this type of bacterium, Desulfovibrio desulfuricans, has 106 cells/mL. These values were used in calculating the concentration of bacteria in the experiments.

2)

Measuring Selectivity of Microorganisms to Mineral Surface

A laser particle size analyzer (FRITSCH Model Analyst 22) was employed for measuring size analysis of single minerals before and after treatment with bacteria. Fixed volume 10 ml of Desulfovibrio desulfuricans solution was conditioned with one gram of each mineral for 30 minutes before recording the change in size distribution, [8, and 10].

3)

Adhesion Measurements

Adhesion of Desulfovibrio desulfuricans bacterial isolate onto the mineral surfaces was determined by dry weight difference before and after conditioning with the mineral particles. 0.5 gram of the ground mineral (‐200 mesh) was added to 80 ml of the cellular suspension with a fixed initial concentration of the bacteria solution, and conditioned for 30 minutes after adjusting the pH values. An additional time of 5 min. was allowed for settling of the mineral particles, after which 20 ml of the supernatant was collected in a porcelain crucible and dried on a hot plate at 40 – 45°C. Adhesion studies were performed as a function of difference in weight before and after drying [8, 10].

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4)

Chemical Analysis

Routine chemical analysis of samples was conducted using standard methods. Phosphorus content was determined using spectroscopic technique. Quartz was determined by Gravimetric method. Meanwhile complete chemical analysis of samples was conducted using ʺPhilipsʺ X‐ray fluorescence (XRF) [8, 10].

5)

Zeta Potential Measurements

A laser Zeta Meter ‘Malvern Instruments Model Zeta Sizer 2000’ was used for zeta potential measurements. 0.01 g of ground sample was placed in 50 ml double distilled water with definite concentration of the bacteria suspension at fixed ionic strength of 2x10‐2 M NaCl. NaOH and HCL were used as pH modifiers. The suspension was conditioned for 30 minutes during which the pH was adjusted. After shaking, the equilibrium pH was recorded. It was then allowed to settle for 3 min, after which 10 ml of the supernatant was transferred into a standard cuvette for zeta potential measurement. Solution temperature was maintained at (25oC ± 2).

Three measurements were taken and the average was reported as the measured zeta potential [8, 10].

6)

Adsorption Studies

The adsorption density of the bacteria on the mineral surface was determined by adding 1 g dry sample of quartz or apatite to the bacteria solutions (50 cm3) in a 100 cm3 volumetric flask. The mixture was shacked for 10 minutes using a shaker (Model JANKE & KUNKEL Type Vx10). The pH was adjusted to the desired values using HCl and NaOH, after which the samples were centrifuged at 15000 rpm for 15 min to separate supernatant from the settled fraction. The total organic carbon content (residual concentration) in the supernatant was determined using a ‘Phoenix 8000’ Total Carbon Analyzerʺ. The average of three readings was taken as a measure for the residual concentration of organic carbon. All the experiments were done at room temperature (25oC ± 2) [8, 10 and 12].

7)

Contact Angle Measurements

The contact angle measurements were carried out through a GONIOMETER RAMÉ–HART using the captive bubble method. A mineral crystal was carefully cut (dimensions at (0.5 × 0.5 × 1.0) cm, and then the surface sample was cautiously polished with a diamond paste containing particles of 3 μm and 1 μm. To remove any particles sticking to the polished surface, the samples were cleaned with jets of distilled water and ultrasonic water bath. The contact angle measurements of the mineral were taken before and after interaction with the bacterial suspension at different pH values. The mineral section was suspended in a bacterial suspension with 10 ml of the biomass for 5 min. After this time, the samples were washed with 10−3 M NaCl solution to remove the bacterial cells that were no stuck. The samples were vacuum dried in desiccators for approximately 10 min.

Finally, the contact angle measurements were carried out. The pH of the solution was adjusted for the same values of the bacterial suspension previously used, using diluted HCl and NaOH solutions 8)

Surface Tension Measurements

The surface tension measurements were performed using the ring method in a Kruss K9 digital TENSIOMETER with an accuracy of ±0.1 mN m−1. The surface tension of the bacterial isolate suspensions was set at different bacterial concentrations and in function of the pH suspension, the pH suspensions were regulated with diluted HCl and NaOH solutions.

9)

Surface Characterization

Selected samples were observed on fractured surface under a JSM‐6400 scanning electron microscope (SEM) to examine the morphology of single mineral before and after treatment with Desulfovibrio desulfuricans.

10) Micro‐Flotation Tests A series of bench‐scale flotation experiments were conducted using a modified Halimond tube with 150 ml capacity. In carrying out these flotation experiments one gram (of single minerals or their binary mixture as well as natural ore) was conditioned at the pre‐determined optimum conditions of pH 9, concentration of bacterial isolate suspension of 10 ml (@106 cells/ mL) and conditioning time 10 minutes using a horizontal shaker. The pH was adjusted with dilute solutions of NaOH and HCl. The flotation was conducted for 5

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minutes at air follow rate of 0.7 cm‐3 /min. Both floated and sink fractions were collected, dried, weighted, and analyzed [8].

Results and Discussions Selectivity of Microorganism to Mineral Surface The change in size distribution of single pure mineral samples, quartz and apatite after its treatment with Desulfovibrio desulfuricans was taken as a measure for the selectivity. Successful adsorption of the bacterial isolate will cause, therefore, a degree of aggregation (or dispersion) for mineral particles leading to a change in their size distribution. This technique was successfully used to screen different microorganisms for selective adhesion onto apatite, quartz or iron oxide surfaces, [8, 10, 11, 13 and 14]. The results obtained in Figures 1 and 2 show different degrees of variation in the size distribution of samples after their treatment with bacterial isolate, which indicates the largest degree of selectivity of Desulfovibrio desulfuricans for apatite more than quartz that caused varying degrees of aggregation for apatite while leaving the quartz particles more dispersing in the pulp.

100 pure apatite d50 = 16.12 mm with MO d50 = 26.66 mm

Cummulatitive wt%

80

60

40

20

0 0.1

1

10

100

Size, m (log)

FIG.2. SIZE DISTRIBUTION OF QUARTZ BEFORE AND AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

FIG.1. SIZE DISTRIBUTION OF APATITE BEFORE AND AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

Surface Properties of Single Minerals and Microorganism The zeta potential measurements of the bacteria alone as well as for each single mineral (quartz–apatite) in absence and presence of Desulfovibrio desulfuricans bacterial isolate have been conducted, Figures 3‐5. Figure 3 depicts that this bacterial isolate is, more or less, hydrophobic in nature where their zeta potential values are varied from about –2 to – 6 mv only over the entire range of pH (pH 2.0 ‐ 12). In the meantime, the values of the zeta potential for the two single minerals were found to be affected by the presence of Desulfovibrio desulfuricans. The surface of each mineral becomes less negative due to the hydrophobic nature of this bacterium.

10

20 apatite apatite with bacteria

2x10-2M NaCl

8

10

0

4

0

2 0 1 -2

2

3

4

5

6

7

8

9

10

11

12

13

14

Zeta, mv

Zeta Potential, mv

6

-10

2

4

6

8

10

12

14

pH

-20

pH -30

-4 -6

-40

-8

-50

FIG.3. ZETA POTENTIAL OF THE DESULFOVIBRIO DESULFURICANS

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FIG.4. ZETA POTENTIAL OF APATITE BEFORE AND AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

Journal of Mining World Express (MWE) Volume 4, 2015 www.mwe‐journal.org

Meanwhile, Figure 4 depicts the zeta potential of apatite as a function of pH before and after interaction with bacteria. These results show that the iso‐electric point (IEP) of apatite corresponds to pH of about (4.8). However, conditioning of apatite with bacteria resulted in a displacement for the IEP of apatite to about (3.7). Moreover, the values of zeta potential of apatite after interaction with bacteria became less negative over the entire pH range. The hydrophobic effect of Desulfovibrio desulfuricans is appeared at pH range (6‐10). On the other hand, conditioning of quartz with Desulfovibrio desulfuricans bacterial isolates resulted in a slight change in the zeta potential values, Figure 5. Interestingly, The values of zeta potential were seen to be shifted to lower values in the entire pH range (1‐11), i.e., the surface of apatite became more hydrophobic and close to those of bacterial isolate of Desulfovibrio desulfuricans as shown before in Figure 4.

75 70

Adhesion%

65 60 55 50 45

quartz apatite

40 35 0

FIG.5. ZETA POTENTIAL OF QUARTZ BEFORE AND AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

2

4

6

8

10

12

pH

FIG.6. ADHESION OF DESULFOVIBRIO DESULFURICANS ONTO MINERALS’ SURFACES

Bacteria Adhesion onto Minerals’ Surfaces Figure 6 shows the adhesion of Desulfovibrio desulfuricans bacteria onto the surface of the two single minerals over a wide range of pH (2‐10). The results showed that, the Desulfovibrio desulfuricans could be adhered onto both minerals’ surfaces with a higher affinity to apatite surface. At the same time, the highest values for adhesion for both quartz and apatite were obtained at pH from 2‐6 followed by a gradual decrease in adhesion values till reaching pH 10.

Surface Tension The surface tension of Desulfovibrio desulfuricans bacterial isolate was conducted at air‐water interface at 25°C±2 and pH 2‐4, Figure 7. The results confirmed a gradual decrease of surface tension values (70‐50 mN/m) by increasing the biomass concentration (1‐10 mL) till reaching a stable state (10‐17 mL).

FIG.7. SURFACE TENSION OF DESULFOVIBRIO DESULFURICANS SUSPENSIONS AT DIFFERENT CONCENTRATIONS

FIG.8. SURFACE TENSION OF DESULFOVIBRIO DESULFURICANS SUSPENSIONS AT DIFFERENT PH VALUES

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On the other hand, the lowest values of surface tension of biomass suspensions of Desulfovibrio desulfuricans bacterial isolate were achieved at pH (1‐4), Figure 8. According to Lambert et al., [15], it was expected that the bacterial isolate suspension could form stable high‐density foam at these pH range. While the foam formed above pH 5 would be less stable.

Contact Angle Measurements Contact angle is a measure of static hydrophobicity. Results obtained through the measurement of contact angles for both single mineral apatite and quartz before and after treatment with Desulfovibrio desulfuricans bacterial isolate confirmed the hydrophobic effect of this type of bacteria. As shown in Figure 9, the values of contact angles as a function of pH are very small (~12° for apatite and ~8° for quartz) which indicated the hydrophilic nature of single minerals’ surfaces. After the interaction with bacterial isolate, there is a significant increase in contact angle values for both apatite and quartz, especially in acidic medium at pH 3 (~35° for apatite and ~15° for quartz). The results also showed that, the Desulfovibrio desulfuricans could be adhered onto both minerals’ surfaces with a higher affinity to apatite surface as mentioned above. The surface of apatite became more hydrophobic than that of quartz due to the adhesion of bacteria cells and/or metabolic products onto the single minerals’ surfaces.

40 apatite + bacterial cell apatite quartz + bacterial cell quartz

Contact angle

30

20

10

0 0

2

4

6

pH

8

10

FIG.9. CONTACT ANGLE OF POLISHED MINERALS CRYSTAL SECTIONS BEFORE AND AFTER INTERACTION WITH DESULFOVIBRIO DESULFURICANS BACTERIA AT A CONCENTRATION ~ 107 CELL/ML

Scanning Electron Microscope (SEM) SEM was used to reveal the morphology and particle size of single minerals before and after treatment with Desulfovibrio desulfuricans. SEM micro‐images indicated that bacterial isolate could be adhered onto both minerals but with higher affinity towards apatite as the amount adhered onto apatite surface is higher than that adhered onto quartz surface, Figures 10 and 11.

Bio‐Flotation of Binary Mixtures Figure 12 shows the effect of pH on adsorption of Desulfovibrio desulfuricans onto quartz and apatite surfaces. The results indicate that the bacterial isolate could be adsorbed onto both minerals surfaces with different degrees where it has a higher affinity to apatite surface. Also, it is noticed that the adsorption density increases at pH from 1‐5 and starts to decrease till reaching pH 11 with the apatite single mineral. On the other hand, the adsorption density increases at pH from 1‐3 and starts to decrease with increasing of pH till reaching pH 11 in case of quartz.

The results of adsorption are in agreement with the results of floatability, Figure 13. The results indicated that the better selectivity could be achieved at pH 3 in which a highest floatability for apatite (~48% floated) and lowest floatability for quartz (~10.21% floated) was obtained after treatment with Desulfovibrio desulfuricans bacterial isolate. The amenability of applying Desulfovibrio desulfuricans to be used as the sole flotation reagent, to selectively separate silica from apatite in their binary mixtures was studied. The results indicated clearly that on using this type of bacterial isolate, a concentrate assaying 98 % P2O5 and 2 % SiO2 with a recovery of 70% could be

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obtained from a mixture containing 90 % by weight apatite and 10% by weight silica. The experiments are performed at pH 3.0 using 1 x 107 cell/ml of bacterial isolate. On applying the same conditions on flotation of the natural phosphate rock, a concentrate containing 30 % P2O5 and 10 % SiO2 with 68 % recovery was obtained from a feed containing 20.52 % P2O5 and 23.51 % SiO2.

FIG.10. SEM OF THE SINGLE APATITE (A) AND (B) AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

FIG.12. EFFECT OF PH ON ADSORPTION OF DESULFOVIBRIO DESULFURICANS ONTO APATITE AND QUARTZ

FIG.11.SEM OF SINGLE QUARTZ (A) AND (B) AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

FIG.13. FLOATABILITY OF SINGLE MINERALS AFTER TREATMENT WITH DESULFOVIBRIO DESULFURICANS

Conclusions The results showed a strong interaction between Desulfovibrio desulfuricans bacterial isolate and minerals’ surfaces, especially with apatite. Adhesion, adsorption, zeta potential measurements showed a better affinity of Desulfovibrio desulfuricans to apatite mineral surface rather than quartz surface.

The results of zeta potential showed that the iso‐electric points (IEP) for apatite mineral (at pH 4.8) is displaced to lower values (at pH 3.7) after interaction with the bacterial isolates, i.e., the IEP became very close to that of Desulfovibrio desulfuricans in which the hydrophobic effect of Desulfovibrio desulfuricans is appeared at pH range (6‐10) .

Higher bacterial affinity to apatite mineral surface in comparison to silica surface is readily evident from the results of adhesion, adsorption, contact angle, surface tension and floatability %.

On using this type of bacterial isolate, a concentrate assaying 98 % P2O5 and 2 % SiO2 with a recovery of 70% could be obtained from a mixture containing 90 % by weight apatite and 10% by weight silica. The experiments are performed at pH 3.0 using 1 x 107 cell/ml of bacterial isolate.

On applying the same conditions on flotation of the natural phosphate rock, a concentrate containing 30 % P2O5 and 10 % SiO2 with 68 % recovery was obtained from a feed containing 20.52 % P2O5 and 23.51 % SiO2 REFERENCES

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