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Effect of fluid shear and particles collision on the oxidation of ferrous iron by Acidithiobacillus ferrooxidans
Minerals Engineering 20 (2007) 1227–1231 This article is also available online at: www.elsevier.com/locate/mineng
Effect of fluid shear and particles collision on the oxidation of ferrous iron by Acidithiobacillus ferrooxidans Guoliang Liu, Junfeng Yin, Wei Cong
*
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Graduate University of the Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 1 November 2006; accepted 16 June 2007 Available online 31 July 2007
Abstract In this paper, the experiments were conducted in a concentric cylinder shear device. The effect on oxidation activity of ferrous iron by Acidithiobacillus ferrooxidans was tested under different shear conditions achieved through regulating the rotating speed of the inner cylinder. The results suggested that the fluid shear rate had little adverse effect on the oxidation activity of At. ferrooxidans. In this study, silicon dioxide powder was added to the medium to investigate the effect of particles collision on the oxidation activity of bacterial cells, and it was found that the oxidation rate of ferrous iron by At. ferrooxidans was greatly decreased with the increase of silicon dioxide concentration. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Bioleaching; Shear forces; Agitation; Bacteria; Oxidation
1. Introduction Bioleaching of sulfide minerals is often carried out in stirred tank reactors for sufficient mass transfer, however, the intensity of shear or turbulence produced by agitation may affect the activity of microorganisms. The shear induced by agitation was addressed as one of the most important rationale behind the limitation of pulp density (Rossi, 2001; Rawlings et al., 2003). Microorganisms had been reported to be highly sensitive to solids concentration and shear conditions with severe limitation in their bioleaching performance at >10% w/v solids (Clark and Norris, 1996; Nemati and Harrison, 2000; Gericke et al., 2001; d’Hugues et al., 2002). It was speculated that the intra-particle shearing in slurry reactors caused a significant decrease in the rate of bioleaching due to the shearing of immobilized microorganisms from the surface of sulfide particles (Karamanev et al., 2001). Effect of agitation rate,
*
Corresponding author. Tel.: +86 10 82627060; fax: +86 10 82627061. E-mail address: [email protected] (W. Cong).
0892-6875/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mineng.2007.06.002
particle size, and shape of solids on the viability of acidophilic bacteria was also examined in stirred tank reactors (Chong et al., 2002; Deveci, 2002, 2004). Most studies on how fluid shear and solids concentration affected the viability of acidophilic bacteria were conducted in stirred tank reactors. However, complex fluid dynamics in the turbulent flow made it very difficult to analyze quantitatively the shear conditions to which the cells were subjected. Quantitative analysis of effects on cells activity under defined shear conditions would be beneficial. A Couette shear device with two concentric cylinders allowed a variable shear rate to be applied in a precise and controlled way (Edwards et al., 1989; Hooker and Lee, 1992). When the inner cylinder rotated, the flow remained laminar only when the speed was fairly low. As the speed increased, the flow was no longer laminar and so-called Taylor flow occurred (Lathrop et al., 1992). Taylor flow was characterized by the existence of rows of alternating toroidal vortices. In Taylor flow, the size of the vortices should be similar to the gap between the cylinders and was usually much larger than the mean droplet size of the particles. Therefore, the emulsion droplets in the Taylor
1228
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
vortices should experience mainly laminar conditions and so it was plausible to treat the stability of colloids in Couette–Taylor flow as analogous to laminar flow (Han et al., 2004). Vaezi et al. (1997) indicated a linear relation between turbulence intensities and average cylinder surface speed and demonstrated the usefulness of the Taylor–Couette apparatus for studies of premixed-flame propagation in high-intensity turbulent flow. In the past years many reports about instability of Taylor–Couette flow had been made (Kim and Choi, 2005; Hayat et al., 2006). In this paper, batch experiments were conducted in a concentric cylinder device set up based on the theory of Couette shear device, for the first time, with the objective to investigate effect of hydrodynamic shear and particles collision on the oxidation rate of ferrous iron by At. ferrooxidans. 2. Materials and methods 2.1. Microorganism and medium The strain of At. ferrooxidans in this study came from Beijing Research Institute of Chemical Engineering and Metallurgy. The culture medium was developed from the 9 K medium by Silverman and Lundgren (1959), which was modified for the growth and the components were as flows: 3 g/L (NH4)2SO4, 0.5 g/L MgSO4 Æ 7H2O, 0.5 g/L K2HPO4, 0.15 g/L K2SO4, 0.01 g/L Ca(NO3)2, and 20 g/L FeSO4 Æ 7H2O. The initial pH value of the medium was adjusted to 1.6 with 6 mol/L H2SO4. The reagents mentioned above were all analytical grade.
water (pH 1.5) and dried in an oven at 50 °C prior to use in the experiments. Based on the theory of Couette shear device a concentric cylinder shear device was designed (Fig. 1), which consisted of two vertical stainless steel concentric cylinders. Both of the cylinders were cone-shaped at the bottom in order to avoid the aggregation of bubbles, which could result in Couette flow turbulence. The dimensions and shape were carefully designed to achieve a uniform shear rate in the experimental region, and equal to that at the sides. The surfaces of the cylinders were polished for increasing experimental accuracy. The outer cylinder remained stationary while the inner cylinder was driven by a DC motor with a DC speed controller. The rotation speed of the inner cylinder was monitored with a digital tachometer by shining at a reflecting flag stuck to the shaft. The temperature of the experimental region maintained constant with water circulating from the water jacket of the shear device to the thermostatic bath. As the gap between the two cylinders was only 5 mm wide, it was not convenient to determine the dissolved oxygen (DO) concentration directly. One circulating system (Fig. 2) branching from the shear device was established for the dissolved oxygen determination with the medium pumped by a peristaltic pump from the experimental region, through the DO probe and back to the shear device. The volume of medium containing in the branch was about 40 ml. As the system was airtight, the determined DO
2.2. Equipment and experimental procedure The bacterial cells suspension was kept at 4 °C and should be activated before use. 25 ml bacteria medium and 100 ml fresh growth medium were added into a 250 ml flat-bottom shake flask, which was put in a thermostatic shaker for about 24 h at 30 °C and 180 rpm. In each batch shear experiment, the initial working volume was 225 ml with 45 ml bacteria medium added to 180 ml fresh medium. The shear experiments in this study when not otherwise stated were conducted at pH 1.6, 30 °C, and 0.15 L air/min. Concentrations of bacterial cells and medium were kept parallel in each batch experiment. The number of bacterial cells was determined by microscopic counting. Sampling of the experiment was performed at predetermined intervals by removing samples with an injector. After each sampling the injector was washed with distilled water and dried at room temperature. The oxidation of ferrous iron was monitored by determining the residual concentration of ferrous iron in the samples, which were titrated with a standardized K2Cr2O7 (Herrera et al., 1989) solution using sodium diphenylamine sulphonate as an indicator. A high-grade silicon dioxide (SiO2) powder (25 lm) was used as inert solids to study the effects of solids concentration ranging from 5% to 20% w/w. The SiO2powder was washed several times with acidified distilled
Fig. 1. The schematic diagram of concentric cylinder shear device.
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
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oxidation process approaching to an end. In the first shear experiment, at the end of 32 h there was a sharper decline of DO concentration than before due to a higher cells concentration and a lower aeration of 0.1 L air/min. The lowest oxygen concentration was 82% at 0.1 L air/min. In the second shear experiment, the DO concentration decreased more slowly due to lower oxidation rate of Fe2+, and the lowest DO concentration was about 90%. It could be inferred that the oxygen supply in the shear device was sufficient for bacteria cells growth and oxidation of ferrous iron. In the flowing experiments, aeration rate was maintained at 0.15 L air/min. Fig. 2. Sketch map of dissolved oxygen determination system.
3.2. Effect of shear rate on oxidation of ferrous iron concentration could approximately reveal the oxygen saturation in the shear device. 3. Results and discussion 3.1. Changes of DO concentration in the oxidation process of Fe2+
100
C Fe2þ ;t¼t0 C Fe2þ ;t¼ti 100%; C Fe2þ ;t¼t0 C Fe2þ ;t¼ti C Fe2þ ;t¼tj t¼ ; tj ti e¼
a
ð2Þ
0.4
95 90 85
125rpm 0% SiO2
80
ð1Þ
where C Fe2þ ;t¼t0 , C Fe2þ ;t¼ti and C Fe2þ ;t¼tj were concentrations of ferrous iron at three different time points, and ti and tj were two successive time points and ti < tj. Fig. 4 shows that the oxidation rates of Fe2+ at various inner cylinder speeds experienced a similar process. The oxidation rate at first increased because of growth of bac-
Fe oxidation rate (g/L/h)
360rpm 15% SiO2
180rpm 360rpm 540rpm 720rpm 900rpm
0.3 0.2 0.1
2+
Oxygen concentration (% saturation)
Two shear experiments were carried out to investigate the changes of oxygen concentration in the oxidation process of Fe2+. In the first shear experiment without SiO2 added, the rotation speed of inner cylinder was maintained at 125 rpm and the aeration was maintained at 0.2 L air/ min at first. At the end of 32 h (indicated at point a in Fig. 3), 60 ml medium was taken out and 80 ml fresh medium was added in shear device. The aeration was changed to 0.1 L air/min at the same time. In the second shear experiment, 15% SiO2 w/w was added, the rotation speed of inner cylinder was maintained at 360 rpm and the aeration was maintained at 0.15 L air/min. Fig. 3 illustrates the changes of DO concentration during the process of oxidation of ferrous iron. The DO concentration decreased with the growth of bacterial cells and oxidation of ferrous iron, and increased with the
The experiments were conducted to investigate how the hydrodynamic shear effect the oxidation of ferrous iron by At. ferrooxidans achieved through maintaining inner cylinder speed at 180, 360, 540, 720, and 900 rpm. It was difficult to differentiate dead bacterial cells from active ones by microscopic counting method. Therefore, conversion ratio (e) and oxidation rate (t) of ferrous iron were used indirectly to describe effects on growth and oxidation activity of bacterial cells under different shear conditions with or without the addition of solids. The equations were as follows:
75
0.0
70 0
10
20
30
40
50
Time (h) Fig. 3. Changes of oxygen concentration during the process of oxidation of ferrous iron in shear device.
0
5
10
15
20
25
Time (h) Fig. 4. Effect of inner cylinder rotation speed on oxidation rate of ferrous iron in the shear device.
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
0.25
100
180rpm 360rpm 540rpm 720rpm 900rpm
80 60
Fe 2+ oxidation rate (g/L/h)
2+
Fe conversion ratio (%)
1230
40 20 0 0
5
10
15
20
5% 10% 15% 20%
0.20 0.15 0.10 0.05
25
Time (h)
0.00 0
Fig. 5. Effect of inner cylinder rotation speed on conversion ratio of ferrous iron in the shear device.
3.3. Effect of SiO2 concentration on the oxidation of ferrous iron Batch experiments were conducted with the SiO2 powder (25 lm) as inert solids to study the effects of solids concentration ranging from 5% to 20% (w/w) with the inner cylinder rotation maintained at 360 rpm. The oxidation rate of ferrous iron tended to decrease with the increase of SiO2 concentration from 5% to 20%, which was illustrated in Fig. 6. The highest oxidation rate was about 0.20 g/(L h) at the solids concentration of 10%, but the highest oxidation rate decreased sharply to below 0.07 g/(L h) when the SiO2 concentration increased to 20%. Fig. 7 demonstrates the conversion ratio of ferrous iron declined with the increase of SiO2 concentration from 5% to 20%. To illustrate, when the oxidation of ferrous iron approached to an end at the SiO2 concentration of
10
15
20
25
30
Time (h) Fig. 6. Effect of SiO2 concentration on oxidation rate of ferrous iron in the shear device at 360 rpm.
100
Fe2+conversion ratio (%)
terial cells, but later it decreased with the oxidation process approaching to an end. Fig. 5 suggests the similar oxidation process of ferrous iron in shear device at different shear rate. It could be inferred that the hydrodynamic shear induced by rotation of inner cylinder in the shear device had little adverse effect on the growth of bacterial cells and oxidation of ferrous iron by At. ferrooxidans. In turbulent flow, the intensity of shear forces acting on microorganisms would probably be related to the relative size of the smallest eddies generated in the system compared with the size of microorganisms. When the size of the smallest eddies approached the size of microorganisms, the adverse shear effects on the cells potentially increased (Doran, 1995; Cherry and Papoutsakis, 1986). In the shear device, as the rotation speed increased, the flow was no longer laminar and Taylor flow occurred. In Taylor flow, the size of the toroidal vortices should be similar to the gap between the cylinders and was usually much larger than the size of bacterial cells (1–1.5 lm) used in current experiments, and therefore, the hydrodynamic shear effects on the bacterial cells would probably be minimal.
5
5% 10% 15% 20%
80 60 40 20 0 0
5
10
15
20
25
30
Time (h) Fig. 7. Effect of SiO2 concentration on conversion ratio of ferrous iron in the shear device at 360 rpm.
5%, the conversion ratio of ferrous iron at the SiO2 concentration of 20% was only about to 40%. As the fluid shear induced by rotation in the shear device had little adverse effect on oxidation of ferrous iron and the dissolved oxygen supply was sufficient, the sharp decrease of the oxidation rate of ferrous iron with the increase of SiO2 concentration probably attributed to the more and more intensive collision and friction between solid particles. The extent of loss in the viability of bacterial population increases as a function of time with increasing the solids density and/or the intensity of agitation (Deveci, 2002). The rate and extent of the deactivation (i.e., loss in viability) of bacterial cells were shown to depend on the impeller type, the size, and shape of solid particles with experiments conducted in stirred tank reactors (Deveci, 2004). Chong et al. (2002) investigated the effect of particle shearing on the rate of sulfide (pyrite) leaching by Thiobacillus ferrooxidans in two very similar bioreactors. These researchers also confirmed the adverse effect on the oxidizing activity induced by collision or shear by solid particles.
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
4. Conclusions In this paper, it was confirmed that the fluid shear rate had little adverse effect on the oxidation activity of bacterial cells. Collision and friction produced by solid particles caused the decrease of oxidation activity of bacterial cells. Therefore, in bioleaching operations suitable solid concentration should be considered in order to reduce the adverse effect on the oxidation activity of bacterial cells. In the design of new bioleaching reactors mixing intensity should be controlled in order to reduce collision and friction of particles resulting in the loss of oxidation activity of bacterial cells. In bioleaching of sulfide minerals, the shear conditions are more complicated than that in the shear device and more detailed experiments need to be conducted to draw firm conclusions. Acknowledgements The authors would like to express his sincere appreciation to the National Basic Research Program (2004CB619203) for the financial support. The authors also wish to thank all the partners of our laboratory. References Cherry, R.S., Papoutsakis, E.T., 1986. Hydrodynamic effects on cells in agitated tissue culture reactors. Bioprocess and Engineering 1, 29–41. Chong, N., Karamanev, D.G., Margaritis, A., 2002. Effect of particle– particle shearing on the bioleaching of sulfide minerals. Biotechnology and Bioengineering 80 (3), 349–357. Clark, D.A., Norris, P.R., 1996. Oxidation of mineral sulphides by thermophilic microorganisms. Minerals Engineering 9 (11), 1119–1125. Deveci, H., 2002. Effect of solids on viability of acidophilic bacteria. Minerals Engineering 15, 1181–1189. Deveci, H., 2004. Effect of particle size and shape of solids on the viability of acidophilic bacteria during mixing in stirred tank reactors. Hydrometallurgy 71 (3–4), 385–396. d’Hugues, P., Foucher, S., Galle-Cavalloni, P., Morin, D., 2002. Continuous bioleaching of chalcopyrite using a novel extremely thermophilic
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mixed culture. International Journal of Mineral Processing 66, 107– 119. Doran, M.P., 1995. Bioprocess Engineering Principles. Academic Press, San Diego. Edwards, N., Beeton, S., Bull, A.T., Merchuk, J.C., 1989. A novel device for the assessment of shear effects on suspended microbial cultures. Applied Microbiology and Biotechnology 30, 190–195. Gericke, M., Pinches, A., van Rooyen, J.V., 2001. Bioleaching of chalcopyrite concentrate using an extremely thermophilic culture. International Journal of Mineral Processing 62, 243–255. Han, J., Washington, C., Melia, C.D., 2004. A concentric cylinder shear device for the study of stability in intravenous emulsions. European Journal of Pharmaceutical Sciences 23, 253–260. Hayat, T., Khan, M., Wang, Y., 2006. Non-Newtonian flow between concentric cylinders. Communications in Nonlinear Science and Numerical Simulation 11 (3), 297–305. Herrera, L., Ruize, P., Aguillon, J.C., Fehrmann, A., 1989. A new spectrophotometric method for the determination of ferrous iron in the presence of ferric iron. Journal of Chemical Technology and Biotechnology 44, 171–181. Hooker, B.S., Lee, J.M., 1992. Application of new structured model to tobacco cell cultures. Biotechnology and Bioengineering 39, 765–774. Karamanev, D.G., Margaritis, A., Chong, N., 2001. The application of ore immobilization to the bioleaching of refractory gold concentrate. International Journal of Mineral Processing 62 (1–4), 231–241. Kim, M.C., Choi, C.K., 2005. The onset of instability in the flow induced by an impulsively started rotating cylinder. Chemical Engineering Science 60 (3), 599–608. Lathrop, D.P., Fineberg, J., Swinney, H.L., 1992. Transition to sheardriven turbulence in Couette–Taylor flow. Physical Review A 46, 6390–6405. Nemati, M., Harrison, S.T.L., 2000. Effect of solid loading on thermophilic bioleaching of sulfide minerals. Journal of Chemical Technology and Biotechnology 74, 562–570. Rawlings, D.E., Dew, D., du Plessis, C., 2003. Biomineralization of metal containing ores and concentrates. Trends in Biotechnology 21 (1), 38– 44. Rossi, G., 2001. The design of bioreactors. Hydrometallurgy 59, 217–231. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Thiobacillus ferrooxidans I: an improved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology 77, 642–647. Vaezi, V., Oh, E.S., Aldredge, R.C., 1997. High-intensity turbulence measurements in a Taylor–Couette flow reactor. Experimental Thermal and Fluid Science 15 (4), 424–431.
Effect of fluid shear and particles collision on the oxidation of ferrous iron by Acidithiobacillus ferrooxidans Guoliang Liu, Junfeng Yin, Wei Cong
*
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Graduate University of the Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China Received 1 November 2006; accepted 16 June 2007 Available online 31 July 2007
Abstract In this paper, the experiments were conducted in a concentric cylinder shear device. The effect on oxidation activity of ferrous iron by Acidithiobacillus ferrooxidans was tested under different shear conditions achieved through regulating the rotating speed of the inner cylinder. The results suggested that the fluid shear rate had little adverse effect on the oxidation activity of At. ferrooxidans. In this study, silicon dioxide powder was added to the medium to investigate the effect of particles collision on the oxidation activity of bacterial cells, and it was found that the oxidation rate of ferrous iron by At. ferrooxidans was greatly decreased with the increase of silicon dioxide concentration. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Bioleaching; Shear forces; Agitation; Bacteria; Oxidation
1. Introduction Bioleaching of sulfide minerals is often carried out in stirred tank reactors for sufficient mass transfer, however, the intensity of shear or turbulence produced by agitation may affect the activity of microorganisms. The shear induced by agitation was addressed as one of the most important rationale behind the limitation of pulp density (Rossi, 2001; Rawlings et al., 2003). Microorganisms had been reported to be highly sensitive to solids concentration and shear conditions with severe limitation in their bioleaching performance at >10% w/v solids (Clark and Norris, 1996; Nemati and Harrison, 2000; Gericke et al., 2001; d’Hugues et al., 2002). It was speculated that the intra-particle shearing in slurry reactors caused a significant decrease in the rate of bioleaching due to the shearing of immobilized microorganisms from the surface of sulfide particles (Karamanev et al., 2001). Effect of agitation rate,
*
Corresponding author. Tel.: +86 10 82627060; fax: +86 10 82627061. E-mail address: [email protected] (W. Cong).
0892-6875/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mineng.2007.06.002
particle size, and shape of solids on the viability of acidophilic bacteria was also examined in stirred tank reactors (Chong et al., 2002; Deveci, 2002, 2004). Most studies on how fluid shear and solids concentration affected the viability of acidophilic bacteria were conducted in stirred tank reactors. However, complex fluid dynamics in the turbulent flow made it very difficult to analyze quantitatively the shear conditions to which the cells were subjected. Quantitative analysis of effects on cells activity under defined shear conditions would be beneficial. A Couette shear device with two concentric cylinders allowed a variable shear rate to be applied in a precise and controlled way (Edwards et al., 1989; Hooker and Lee, 1992). When the inner cylinder rotated, the flow remained laminar only when the speed was fairly low. As the speed increased, the flow was no longer laminar and so-called Taylor flow occurred (Lathrop et al., 1992). Taylor flow was characterized by the existence of rows of alternating toroidal vortices. In Taylor flow, the size of the vortices should be similar to the gap between the cylinders and was usually much larger than the mean droplet size of the particles. Therefore, the emulsion droplets in the Taylor
1228
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
vortices should experience mainly laminar conditions and so it was plausible to treat the stability of colloids in Couette–Taylor flow as analogous to laminar flow (Han et al., 2004). Vaezi et al. (1997) indicated a linear relation between turbulence intensities and average cylinder surface speed and demonstrated the usefulness of the Taylor–Couette apparatus for studies of premixed-flame propagation in high-intensity turbulent flow. In the past years many reports about instability of Taylor–Couette flow had been made (Kim and Choi, 2005; Hayat et al., 2006). In this paper, batch experiments were conducted in a concentric cylinder device set up based on the theory of Couette shear device, for the first time, with the objective to investigate effect of hydrodynamic shear and particles collision on the oxidation rate of ferrous iron by At. ferrooxidans. 2. Materials and methods 2.1. Microorganism and medium The strain of At. ferrooxidans in this study came from Beijing Research Institute of Chemical Engineering and Metallurgy. The culture medium was developed from the 9 K medium by Silverman and Lundgren (1959), which was modified for the growth and the components were as flows: 3 g/L (NH4)2SO4, 0.5 g/L MgSO4 Æ 7H2O, 0.5 g/L K2HPO4, 0.15 g/L K2SO4, 0.01 g/L Ca(NO3)2, and 20 g/L FeSO4 Æ 7H2O. The initial pH value of the medium was adjusted to 1.6 with 6 mol/L H2SO4. The reagents mentioned above were all analytical grade.
water (pH 1.5) and dried in an oven at 50 °C prior to use in the experiments. Based on the theory of Couette shear device a concentric cylinder shear device was designed (Fig. 1), which consisted of two vertical stainless steel concentric cylinders. Both of the cylinders were cone-shaped at the bottom in order to avoid the aggregation of bubbles, which could result in Couette flow turbulence. The dimensions and shape were carefully designed to achieve a uniform shear rate in the experimental region, and equal to that at the sides. The surfaces of the cylinders were polished for increasing experimental accuracy. The outer cylinder remained stationary while the inner cylinder was driven by a DC motor with a DC speed controller. The rotation speed of the inner cylinder was monitored with a digital tachometer by shining at a reflecting flag stuck to the shaft. The temperature of the experimental region maintained constant with water circulating from the water jacket of the shear device to the thermostatic bath. As the gap between the two cylinders was only 5 mm wide, it was not convenient to determine the dissolved oxygen (DO) concentration directly. One circulating system (Fig. 2) branching from the shear device was established for the dissolved oxygen determination with the medium pumped by a peristaltic pump from the experimental region, through the DO probe and back to the shear device. The volume of medium containing in the branch was about 40 ml. As the system was airtight, the determined DO
2.2. Equipment and experimental procedure The bacterial cells suspension was kept at 4 °C and should be activated before use. 25 ml bacteria medium and 100 ml fresh growth medium were added into a 250 ml flat-bottom shake flask, which was put in a thermostatic shaker for about 24 h at 30 °C and 180 rpm. In each batch shear experiment, the initial working volume was 225 ml with 45 ml bacteria medium added to 180 ml fresh medium. The shear experiments in this study when not otherwise stated were conducted at pH 1.6, 30 °C, and 0.15 L air/min. Concentrations of bacterial cells and medium were kept parallel in each batch experiment. The number of bacterial cells was determined by microscopic counting. Sampling of the experiment was performed at predetermined intervals by removing samples with an injector. After each sampling the injector was washed with distilled water and dried at room temperature. The oxidation of ferrous iron was monitored by determining the residual concentration of ferrous iron in the samples, which were titrated with a standardized K2Cr2O7 (Herrera et al., 1989) solution using sodium diphenylamine sulphonate as an indicator. A high-grade silicon dioxide (SiO2) powder (25 lm) was used as inert solids to study the effects of solids concentration ranging from 5% to 20% w/w. The SiO2powder was washed several times with acidified distilled
Fig. 1. The schematic diagram of concentric cylinder shear device.
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
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oxidation process approaching to an end. In the first shear experiment, at the end of 32 h there was a sharper decline of DO concentration than before due to a higher cells concentration and a lower aeration of 0.1 L air/min. The lowest oxygen concentration was 82% at 0.1 L air/min. In the second shear experiment, the DO concentration decreased more slowly due to lower oxidation rate of Fe2+, and the lowest DO concentration was about 90%. It could be inferred that the oxygen supply in the shear device was sufficient for bacteria cells growth and oxidation of ferrous iron. In the flowing experiments, aeration rate was maintained at 0.15 L air/min. Fig. 2. Sketch map of dissolved oxygen determination system.
3.2. Effect of shear rate on oxidation of ferrous iron concentration could approximately reveal the oxygen saturation in the shear device. 3. Results and discussion 3.1. Changes of DO concentration in the oxidation process of Fe2+
100
C Fe2þ ;t¼t0 C Fe2þ ;t¼ti 100%; C Fe2þ ;t¼t0 C Fe2þ ;t¼ti C Fe2þ ;t¼tj t¼ ; tj ti e¼
a
ð2Þ
0.4
95 90 85
125rpm 0% SiO2
80
ð1Þ
where C Fe2þ ;t¼t0 , C Fe2þ ;t¼ti and C Fe2þ ;t¼tj were concentrations of ferrous iron at three different time points, and ti and tj were two successive time points and ti < tj. Fig. 4 shows that the oxidation rates of Fe2+ at various inner cylinder speeds experienced a similar process. The oxidation rate at first increased because of growth of bac-
Fe oxidation rate (g/L/h)
360rpm 15% SiO2
180rpm 360rpm 540rpm 720rpm 900rpm
0.3 0.2 0.1
2+
Oxygen concentration (% saturation)
Two shear experiments were carried out to investigate the changes of oxygen concentration in the oxidation process of Fe2+. In the first shear experiment without SiO2 added, the rotation speed of inner cylinder was maintained at 125 rpm and the aeration was maintained at 0.2 L air/ min at first. At the end of 32 h (indicated at point a in Fig. 3), 60 ml medium was taken out and 80 ml fresh medium was added in shear device. The aeration was changed to 0.1 L air/min at the same time. In the second shear experiment, 15% SiO2 w/w was added, the rotation speed of inner cylinder was maintained at 360 rpm and the aeration was maintained at 0.15 L air/min. Fig. 3 illustrates the changes of DO concentration during the process of oxidation of ferrous iron. The DO concentration decreased with the growth of bacterial cells and oxidation of ferrous iron, and increased with the
The experiments were conducted to investigate how the hydrodynamic shear effect the oxidation of ferrous iron by At. ferrooxidans achieved through maintaining inner cylinder speed at 180, 360, 540, 720, and 900 rpm. It was difficult to differentiate dead bacterial cells from active ones by microscopic counting method. Therefore, conversion ratio (e) and oxidation rate (t) of ferrous iron were used indirectly to describe effects on growth and oxidation activity of bacterial cells under different shear conditions with or without the addition of solids. The equations were as follows:
75
0.0
70 0
10
20
30
40
50
Time (h) Fig. 3. Changes of oxygen concentration during the process of oxidation of ferrous iron in shear device.
0
5
10
15
20
25
Time (h) Fig. 4. Effect of inner cylinder rotation speed on oxidation rate of ferrous iron in the shear device.
G. Liu et al. / Minerals Engineering 20 (2007) 1227–1231
0.25
100
180rpm 360rpm 540rpm 720rpm 900rpm
80 60
Fe 2+ oxidation rate (g/L/h)
2+
Fe conversion ratio (%)
1230
40 20 0 0
5
10
15
20
5% 10% 15% 20%
0.20 0.15 0.10 0.05
25
Time (h)
0.00 0
Fig. 5. Effect of inner cylinder rotation speed on conversion ratio of ferrous iron in the shear device.
3.3. Effect of SiO2 concentration on the oxidation of ferrous iron Batch experiments were conducted with the SiO2 powder (25 lm) as inert solids to study the effects of solids concentration ranging from 5% to 20% (w/w) with the inner cylinder rotation maintained at 360 rpm. The oxidation rate of ferrous iron tended to decrease with the increase of SiO2 concentration from 5% to 20%, which was illustrated in Fig. 6. The highest oxidation rate was about 0.20 g/(L h) at the solids concentration of 10%, but the highest oxidation rate decreased sharply to below 0.07 g/(L h) when the SiO2 concentration increased to 20%. Fig. 7 demonstrates the conversion ratio of ferrous iron declined with the increase of SiO2 concentration from 5% to 20%. To illustrate, when the oxidation of ferrous iron approached to an end at the SiO2 concentration of
10
15
20
25
30
Time (h) Fig. 6. Effect of SiO2 concentration on oxidation rate of ferrous iron in the shear device at 360 rpm.
100
Fe2+conversion ratio (%)
terial cells, but later it decreased with the oxidation process approaching to an end. Fig. 5 suggests the similar oxidation process of ferrous iron in shear device at different shear rate. It could be inferred that the hydrodynamic shear induced by rotation of inner cylinder in the shear device had little adverse effect on the growth of bacterial cells and oxidation of ferrous iron by At. ferrooxidans. In turbulent flow, the intensity of shear forces acting on microorganisms would probably be related to the relative size of the smallest eddies generated in the system compared with the size of microorganisms. When the size of the smallest eddies approached the size of microorganisms, the adverse shear effects on the cells potentially increased (Doran, 1995; Cherry and Papoutsakis, 1986). In the shear device, as the rotation speed increased, the flow was no longer laminar and Taylor flow occurred. In Taylor flow, the size of the toroidal vortices should be similar to the gap between the cylinders and was usually much larger than the size of bacterial cells (1–1.5 lm) used in current experiments, and therefore, the hydrodynamic shear effects on the bacterial cells would probably be minimal.
5
5% 10% 15% 20%
80 60 40 20 0 0
5
10
15
20
25
30
Time (h) Fig. 7. Effect of SiO2 concentration on conversion ratio of ferrous iron in the shear device at 360 rpm.
5%, the conversion ratio of ferrous iron at the SiO2 concentration of 20% was only about to 40%. As the fluid shear induced by rotation in the shear device had little adverse effect on oxidation of ferrous iron and the dissolved oxygen supply was sufficient, the sharp decrease of the oxidation rate of ferrous iron with the increase of SiO2 concentration probably attributed to the more and more intensive collision and friction between solid particles. The extent of loss in the viability of bacterial population increases as a function of time with increasing the solids density and/or the intensity of agitation (Deveci, 2002). The rate and extent of the deactivation (i.e., loss in viability) of bacterial cells were shown to depend on the impeller type, the size, and shape of solid particles with experiments conducted in stirred tank reactors (Deveci, 2004). Chong et al. (2002) investigated the effect of particle shearing on the rate of sulfide (pyrite) leaching by Thiobacillus ferrooxidans in two very similar bioreactors. These researchers also confirmed the adverse effect on the oxidizing activity induced by collision or shear by solid particles.
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4. Conclusions In this paper, it was confirmed that the fluid shear rate had little adverse effect on the oxidation activity of bacterial cells. Collision and friction produced by solid particles caused the decrease of oxidation activity of bacterial cells. Therefore, in bioleaching operations suitable solid concentration should be considered in order to reduce the adverse effect on the oxidation activity of bacterial cells. In the design of new bioleaching reactors mixing intensity should be controlled in order to reduce collision and friction of particles resulting in the loss of oxidation activity of bacterial cells. In bioleaching of sulfide minerals, the shear conditions are more complicated than that in the shear device and more detailed experiments need to be conducted to draw firm conclusions. Acknowledgements The authors would like to express his sincere appreciation to the National Basic Research Program (2004CB619203) for the financial support. The authors also wish to thank all the partners of our laboratory. References Cherry, R.S., Papoutsakis, E.T., 1986. Hydrodynamic effects on cells in agitated tissue culture reactors. Bioprocess and Engineering 1, 29–41. Chong, N., Karamanev, D.G., Margaritis, A., 2002. Effect of particle– particle shearing on the bioleaching of sulfide minerals. Biotechnology and Bioengineering 80 (3), 349–357. Clark, D.A., Norris, P.R., 1996. Oxidation of mineral sulphides by thermophilic microorganisms. Minerals Engineering 9 (11), 1119–1125. Deveci, H., 2002. Effect of solids on viability of acidophilic bacteria. Minerals Engineering 15, 1181–1189. Deveci, H., 2004. Effect of particle size and shape of solids on the viability of acidophilic bacteria during mixing in stirred tank reactors. Hydrometallurgy 71 (3–4), 385–396. d’Hugues, P., Foucher, S., Galle-Cavalloni, P., Morin, D., 2002. Continuous bioleaching of chalcopyrite using a novel extremely thermophilic
1231
mixed culture. International Journal of Mineral Processing 66, 107– 119. Doran, M.P., 1995. Bioprocess Engineering Principles. Academic Press, San Diego. Edwards, N., Beeton, S., Bull, A.T., Merchuk, J.C., 1989. A novel device for the assessment of shear effects on suspended microbial cultures. Applied Microbiology and Biotechnology 30, 190–195. Gericke, M., Pinches, A., van Rooyen, J.V., 2001. Bioleaching of chalcopyrite concentrate using an extremely thermophilic culture. International Journal of Mineral Processing 62, 243–255. Han, J., Washington, C., Melia, C.D., 2004. A concentric cylinder shear device for the study of stability in intravenous emulsions. European Journal of Pharmaceutical Sciences 23, 253–260. Hayat, T., Khan, M., Wang, Y., 2006. Non-Newtonian flow between concentric cylinders. Communications in Nonlinear Science and Numerical Simulation 11 (3), 297–305. Herrera, L., Ruize, P., Aguillon, J.C., Fehrmann, A., 1989. A new spectrophotometric method for the determination of ferrous iron in the presence of ferric iron. Journal of Chemical Technology and Biotechnology 44, 171–181. Hooker, B.S., Lee, J.M., 1992. Application of new structured model to tobacco cell cultures. Biotechnology and Bioengineering 39, 765–774. Karamanev, D.G., Margaritis, A., Chong, N., 2001. The application of ore immobilization to the bioleaching of refractory gold concentrate. International Journal of Mineral Processing 62 (1–4), 231–241. Kim, M.C., Choi, C.K., 2005. The onset of instability in the flow induced by an impulsively started rotating cylinder. Chemical Engineering Science 60 (3), 599–608. Lathrop, D.P., Fineberg, J., Swinney, H.L., 1992. Transition to sheardriven turbulence in Couette–Taylor flow. Physical Review A 46, 6390–6405. Nemati, M., Harrison, S.T.L., 2000. Effect of solid loading on thermophilic bioleaching of sulfide minerals. Journal of Chemical Technology and Biotechnology 74, 562–570. Rawlings, D.E., Dew, D., du Plessis, C., 2003. Biomineralization of metal containing ores and concentrates. Trends in Biotechnology 21 (1), 38– 44. Rossi, G., 2001. The design of bioreactors. Hydrometallurgy 59, 217–231. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Thiobacillus ferrooxidans I: an improved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology 77, 642–647. Vaezi, V., Oh, E.S., Aldredge, R.C., 1997. High-intensity turbulence measurements in a Taylor–Couette flow reactor. Experimental Thermal and Fluid Science 15 (4), 424–431.