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Effects of dissolved oxygen on the biooxidation process of refractory gold ores
Journal of Bioscience and Bioengineering VOL. 114 No. 5, 531e536, 2012 www.elsevier.com/locate/jbiosc
Effects of dissolved oxygen on the biooxidation process of refractory gold ores Li-Xin Sun, Xu Zhang,* Wen-Song Tan, and Ming-Long Zhu The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China Received 18 April 2012; accepted 7 June 2012 Available online 9 August 2012
While multiple theories exist regarding the effect of dissolved oxygen (DO) on the biooxidation of minerals, few studies have been performed the cellular or molecular scale (e.g., genetics) and the mechanism remains unclear. In this paper, the effects of DO concentration on the biooxidation process of refractory sulfide gold ores by Acidithiobacillus ferrooxidans were investigated in the experimental stirred tank bioreactors (STRs). The results indicated that higher biooxidation and cell growth rates were correlated with higher DO concentration. The biooxidation process was restricted at 1.2 ppm DO due to oxygen limitation. Furthermore, the effects of DO on cellular and molecular scale were studied for the first time. The results demonstrated that the oxygen uptake rate (OUR), the Fe2D oxidation activity and the rus gene expression of A. ferrooxidans all increased with the DO concentration, which might be responsible for the increase of the biooxidation rates with the DO concentration. This study provides insight into the potential impact of molecular-level mechanisms of DO in the biooxidation process of minerals. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Refractory sulfide gold ores; Biooxidation process; Dissolved oxygen; Acidithiobacillus ferrooxidans; Stirred tank bioreactors]
Biooxidation of sulfidic-refractory gold minerals is often applied in the pretreatment of refractory sulfide gold ores. Currently, the commercial process of the biooxidation had been successfully used in the gold processing (1). Acidithiobacillus ferrooxidans, one of the primary organisms in the commercial biooxidation industries, is an acidophilic chemolithotrophic gram-negative bacterium. A. ferrooxidans obtains energy by the oxidation of ferrous ions to ferric ions and uses molecular oxygen as the terminal electron acceptor in aerobic conditions (2). Therefore, the dissolved oxygen (DO) plays an important role in the biooxidation process of refractory gold ores. Previous reports had indicated that a minimum concentration of DO (1.2e1.5 ppm) was required to avoid the limitation (3,4) (Dew, D. W and Miller, G., Abstr. IBS97, Aus., p. M7.1.1-M7.1.9, 1997). Additionally, Liu et al. (5) found that A. ferrooxidans did not grow in 9K medium, if the DO concentration was below 0.20 mg/L. Gericke et al. (6) claimed that the DO concentration limitation was 50% during the bioleaching of chalcopyrite concentrate using an extremely thermophilic culture. Furthermore, it had been found that both the cell specific growth rate and oxidation rate increased with the oxygen concentration (7,8). In order to ensure the DO concentration is maintained above limitation concentration, the oxygen mass transfer rate was often heightened by increasing either aeration or agitation intensity. However, these options would result in additional energy
consumption. Conversely, they may inhibit the microbial growth and biooxidation rates if, for example, the agitation intensity is beyond a certain level (4). To date, few studies have been performed on the effects of DO at a cellular or molecular scale (e.g., genetics), which limited the understanding of the mechanism and the potential optimization methods for the biooxidation process. To address this need, in this work, the effects of DO on the biooxidation rates, the activity of bacterial cells, the Fe2þ oxidation activity and the expression levels of rus genes of A. ferrooxidans were investigated in the experimental STRs. The results of this study were expected to further the current understanding of the effects of the DO concentration on the biooxidation process and provide scientific guidance for the design and optimization of the biooxidation process.
* Corresponding author at: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, P. O. Box 309, Shanghai 200237, PR China. Tel.: þ86 21 64252536; fax: þ86 21 64252250. E-mail address: [email protected] (X. Zhang).
Experimental procedures The batch biooxidation experiments were carried out in three 1.5 L STRs with 900 ml of iron-free 9K medium, 100 ml of the inoculated bacterial culture and 100 g of ores as energy source. The agitation was provided by a six-bladed 45 downward pitched blade turbine (PBT) impeller. A sparger was
MATERIALS AND METHODS Sample The low-grade refractory gold ores used in the experiments were kindly supplied by Tiancheng Co., Ltd., Shandong, P. R. China, containing 24.9 g/t Au, 96.1 g/t Ag, 21.30% Fe, 20.09% S, 7.74% C, 4.59% As, 0.02% Cu, 0.31% Pb and 0.13% Zn. Xray diffraction (XRD) analysis showed that the major components of the sample was pyrite and quartz. Microorganism and culture medium The strain of A. provided by Tiancheng Co., Ltd. The culture medium (iron-free developed from the 9K medium (9). The composition was (NH4)2SO4, 3.0; KCl, 0.1; K2HPO4, 0.5; MgSO4$7H2O, 0.5; Ca chemical reagents were of analytical grade.
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.06.004
ferrooxidans was 9K medium) was as follows (g/L): (NO3)2, 0.01. All
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situated below the bottom impeller. The dry air aeration rate was measured by a gas rotameter and controlled by a valve attached to the rotameter. The aeration rate was adjusted from 0.02 to 2 L/min, to stabilize the DO concentration at 5.2 ppm, 3.1 ppm or 1.2 ppm (20%, 50% and 80% of the saturation value, respectively) during the biooxidation process. The environmental conditions were as follows: 41 C, pH 1.5, the impeller speed in the STRs was 300 rpm. Analytical methods The pH and the DO concentration of the suspension were measured by a pH meter (Mettler model FE20) and a DO meter (OXYFERM 225, Hamilton). The mineral samples were collected after biooxidation for SEM (JEOL JSM-6360 LV) analysis. Mineral dissolution was determined by measuring the iron concentration in solution. The concentration of ferrous iron was determined by titration with potassium dichromate in the presence of the indicator N-phenylanthranilic acid (10). The ferric iron concentration was determined by titration EDTA at pH 2 in the presence of the indicator sulphosalicylic acid (11). Total iron concentration in the liquid phase was the summation of the ferric and ferrous iron concentrations. The OUR was measured using the dynamic gas-out/gas-in method (12). The number of free cells in solution was measured by direct microscopic counting using a Pettrof-Hauser-type cell counter. The ores oxidation ratio (l) and oxidation rate (y) were calculated as follows (8):
l ¼
y ¼
½Feti ½Feto ½Fep ½Fetj ½Feti tj ti
! 100%
(1)
(2)
where ½Feti and ½Fetj are the iron concentrations in the liquid at two consecutive time points, respectively. ½Fet0 and [Fe]p are the iron concentrations in the liquid and in the ores, respectively, prior to biooxidation. All experiments were performed in triplicate to reduce the likelihood that our findings were taking place by chance. The Fe2þ oxidation activity of A. ferrooxidans at Fe2D oxidation activity different DO concentrations was determined by measuring the concentration of oxidized Fe2þ in the reaction mixture under aerobic conditions (13). The reaction mixture was composed of 2 ml 0.1 M alanine-SO24 buffer (pH 3.0), washed intact bacterial cells, and 1 ml ferrous sulphate (CFe2þ ¼ 0:2 g=L). The total volume of the mixture was 3.0 ml. The reaction was carried out by shaking the reaction mixture at 41 C. A sample of the reaction mixture (0.2 ml) was withdrawn every 15 s. The concentration of Fe2þ was then determined spectrophotometrically using the o-phenanthroline method (14). Protein concentration in the mixture was measured with the Bradford method (15), using crystalline bovine serum albumin as the reference protein. The Fe2þ oxidation activity was then defined as the decrease of ferrous ion concentration per second per gram of dry cell mass.
Quantitative real-time PCR The relative expression levels of the rus gene of A. ferrooxidans grew at different DO concentrations was determined with real-time PCR, using the Mx3000 Real-Time PCR System (Stratagene, La Jolla, CA, USA). The cell were collected during the exponential phase by filtration of the liquid through a 0.22 mm pore-size membrane. Then, the total cellular RNA was extracted using the TRIzol reagent (TianGen Biotech, Beijing, P.R. China) according to the manufacturer’s instructions. The synthesis of cDNA from RNA samples was performed using the 1st Strand cDNA synthesis kit (MBI, USA), according to the manufacturer’s instructions. These reaction mixtures were incubated at 50 C for 30 min, and the reaction was terminated by heating the samples at 85 C for 5 min. Quantitative RT-PCRs of the resultant cDNA were performed with the Mx3000 Real-Time PCR System and the Maxima SYBR Green q-PCR Master Mix Kit (MBI, America). The reaction mixture contained 12.5 ml of SYBRÒ Green PCR Master Mix (Biotools), 1 mL of cDNA, 1 mL of the corresponding, and 0.5 ml of each primer (detailed below). The mixture was brought 25 ml of total volume with nuclease-free water. The amplification program consisted of 1 cycle at 95 C for 5 min, and then 40 cycles at 95 C for 30 s, 58 C for 60 s and 72 C for 60 s. The real-time PCR primers were 50 -CGCGATGGCCGGTACTCTGGA-30 and 50 -CCGCCGCGACCACATGTACAGT-30 for the rus gene, 5-AATCCAAGAAGAAGCACCG-30 and 50 -CCACTGATGTTCCTCCAG-30 for the 16S rRNA (a house-keeping gene, as a control). The threshold cycle (Ct) values were determined using the curve analytical software of the Mx3000 system (Stratagene). The fold change in the target gene was determined by the formula 2DDCt (16), where DDCt ¼ (DCt from the experimental condition) (DCt from control). The DCt was calculated as the difference between the Ct of the target gene and Ct of the reference gene (16S rRNA).
RESULTS AND DISCUSSION Effects of DO on the mineral oxidation The concentration of Fe2þ and Fe3þ in solution, the ores oxidation ratio and the biooxidation rate during the biooxidation process at various DO concentrations are shown in Fig. 1. As shown in Fig. 1AeC, the concentration of Fe3þ and the oxidation ratio at 1.2 ppm DO were lower than those at 5.2 ppm DO. However, the concentration of Fe2þ was higher at 1.2 ppm DO than at 5.2 ppm DO. It should be noted that the biooxidation process was inhibited at 1.2 ppm DO due to oxygen limitation. The concentration of oxygen limitation was closed to 1.5 ppm which was proposed by Dew and Miller (Dew, D. W. and Miller, G., Abstr. IBS97, Aus., p. M7.1.1-M7.1.9, 1997).
FIG. 1. Evolutions of the concentration of Fe2þ (A) and Fe3þ (B), the ores oxidation ratio (C) and rate (D) at various DO concentrations.
VOL. 114, 2012 It illustrated from Fig. 1D that the oxidation rate increased with the growth process during the first 160 h and then decreased. Conversely, the oxidation rate increased with the DO concentration. The highest observed biooxidation rate was about 0.087 g/L/h at 5.2 ppm DO, but decreased to 0.057 g/L/h at 1.2 ppm DO. The biooxidation rate at 5.2 ppm was only slightly higher than the rate at 3.1 ppm. As the terminal electron acceptor, the availability of the DO was observed to be the limiting factor during Fe2þ oxidation by A. ferrooxidans. Therefore, adequate DO availability could accelerate the process of Fe2þ oxidization to Fe3þ. These results agreed well with the found of Gleisner et al. (8). The SEM micrographs of the oxidized mineral are shown in Fig. 2AeE. These micrographs illustrate that while the mineral surface was smooth prior to the biooxidation process (Fig. 2A), but became rough after the biooxidation process (Fig. 2BeD). As displayed in Fig. 2B and C, there were obviously several holes of approximately 1e2 mm in length on the mineral surface. These holes were not observed under abiotic conditions. Instead, irregular long and narrow cracks were observed on the mineral surface before biooxidation (Fig. 2E). A similar observation had been reported in previous studies (17e20). It was hypothesized that the
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holes on the mineral surface were caused by the adhesion of bacterial cells, and the cracks developed from initial mineral defects. These corrosion pits were all conducive to the mineral oxidation. Effects of DO on the suspended cells growth and the activity of bacterial The effect of DO concentration on the growth of suspended bacterial cells during the biooxidation process is shown in Fig. 3. At 5.2 ppm DO, the cells grew markedly during the exponential growth phase without a lag phase, and the growth process reached the stationary phase after 138 h. However, at 3.1 ppm and 1.2 ppm DO, the concentration of suspended cells decreased during the first 24 h due to the adhesion of the cells onto the mineral surface. The exponential growth periods were 156 h at 3.1 ppm DO and 186 h at 1.2 ppm DO. These results showed that the exponential growth phase of A. ferrooxidans was shorter at higher DO concentrations than at lower DO concentrations. Thus, it could hypothesize that the cells’s growth was restrained at lower DO concentration. However, eventually all three experimental groups reached the same final concentrations, approximately 1.6 108 cell/ml. It was different from the results of Gleisner et al. (8) that the concentration of
FIG. 2. The SEM micrographs of mineral surface before (A) and after biooxidated by A. ferrooxidans at 5.2 ppm DO (B), 3.1 ppm DO (C), 1.2 ppm DO (D) and abiotic (E) as control.
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FIG. 5. The Fe2þ oxidation activity of A. ferrooxidans during the exponential growth phase at various DO concentrations. FIG. 3. Evolutions of the suspended cell concentration at various DO concentrations.
A. ferrooxidans generally increased with the DO concentration. However, in that study, the final concentration of cells was 1e20 106 cell/ml, which was one order of magnitude smaller in number that those used in this study. Thus, the difference in the cell concentration might be responsible for differences in the results. The oxygen uptake rate (OUR) which could provide important information about the metabolic activity of the cultured cells, has been previously used for optimizing the fermentation process (21). The OUR of bacteria is based on the presumption that only the active cells will oxidize ferrous iron and hence consume oxygen (22). Fig. 4 shows the relationship between DO concentration and the OUR of A. ferrooxidans during the biooxidation process. It could be seen that OUR increased during the bacterial exponential growth phase, reached the maximum in the late period of cell growth and subsequently decreased. Because the Fe2þ oxidization rate increased as the cells grew in the exponential growth phase, the OUR also initially increased. Then, as the cells began to die and the overall metabolic activity decreased, the OUR also decreased. In addition, the OUR was greater at higher DO concentrations. The maximum OUR was 0.052 mg/L/s at 5.2 ppm DO, with a sharp decrease to 0.034 mg/L/s at 1.2 ppm DO. This finding indicated that the activity of the bacterial population increased with the DO concentration.
FIG. 4. Evolution of the OUR during the biooxidation at various DO concentrations.
Effect of DO on the Fe2D oxidation activity of A. ferrooxidans There is a positive correlation between the biomass concentration and the observed OUR, which is intrinsically linked in the activity of bacterial population (23). However, the OUR could not directly reflect the oxidation activity of any individual cell. The Fe2þ oxidation activity of A. ferrooxidans during the exponential growth phase was determined in this paper and is shown in Fig. 5. The results indicated that the Fe2þ oxidation activity increased with the DO concentration, with activity observed to be w 25% higher at 5.2 ppm DO than that at 1.2 ppm DO. In aerobic conditions, A. ferrooxidans obtains energy from the oxidation process of ferrous ions to ferric ions using molecular oxygen as the terminal electron acceptor in the respiratory chain (2). Additionally, the cells also get reducing power derived from the respiratory chain. Both of them are necessary for many subsequent metabolic processes, including CO2 fixation (24). The Fe2þ oxidation activity of A. ferrooxidans could reflect both the electron transport rate of respiratory chain and the oxidation activity of the individual cell. The higher Fe2þ oxidation activity, the higher electron transport rate of respiratory chain. Namely, bacteria may get more energy and reducing power at higher DO concentration, resulting in an increase in the metabolic activity of the cells. Effect of DO on expression of rus genes of A. ferrooxidans Several redox proteins involved in the electron transfer process have been isolated and characterized in A. ferrooxidans. Among these, the 16.5-kDa periplasmic copper protein rusticyanin (encoded by the rus gene), have received special attention because it may constitute as much as 5% of the total soluble proteins in A. ferrooxidans during autotrophic growth on iron (25). It had been suggested that rusticyanin functioned as an electron reservoir by readily took up electrons available at the outer membrane and then channelled them through the respiratory pathway. The study of Yarzábal et al. (26) showed that the iron oxidation took place only when the rusticyanin had reached a critical intracellular level. Liu et al. (27) also found that by overexpressing the rus gene, it was possible to increase the Fe2þ oxidation activity. In order to study the effect of DO concentration on the Fe2þ oxidation activity, the effect of DO concentration on the rus gene expression of A. ferrooxidans was determined. The relative transcription levels of the rus gene at the three DO concentrations are shown in Fig. 6. It was demonstrated that the expression levels of the rus gene of A. ferrooxidans also increased with DO concentration. The rus gene
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expression. However, future studies will be required to confirm this hypothesis. ACKNOWLEDGMENTS This study was financially supported by Open Project Funding of State Key Laboratory of Bioreactor Engineering of China and the National High Technology Research and Development Program of China (Nos. 2007AA060904 and 2012AA061503). References
FIG. 6. Effect of DO concentration on the expression of the rus gene during the biooxidation of A. ferrooxidans (rus gene expression in 1.2 ppm DO condition was designated as control).
expression was restrained at 1.2 ppm DO, which might lead to a lack of intracellular rusticyanin in A. ferrooxidans. This lack of rusticyanin might have ultimately resulted in the observed decrease of the Fe2þ oxidation activity and the restriction of the biooxidation process at 1.2 ppm DO. It could be found from Figs. 5 and 6, respectively, that the rus gene of A. ferrooxidans increased 60% and the Fe2þ oxidation activity increased 22%, when the DO concentration increased from 1.2 ppm to 3.1 ppm. Thus, increased the rus gene expression was an effective way to improve the Fe2 þ oxidation activity. It can be hypothesized that, if the rus gene expression of A. ferrooxidans could be upregulated artificially at the lower DO concentration, the Fe2þ oxidation activity cells would increase, thereby accelerating the rate of ore biooxidation. The research of Liu et al. (27) provided such an approach by overexpressing recombinant rus gene products in an engineered strain of A. ferrooxidans (pTRUS) containing a homologous rus gene. However, this method could achieve the desired goals at lower DO concentrations. Future studies will need to be conducted to test if this is the case. However, the expression levels of the rus gene increased by 130%, but Fe2 þ oxidation activity increased by only 4%, when the DO concentration increased from 3.1 ppm to 5.2 ppm. Therefore, it appeared that the increase in DO concentration could not improve the Fe2þ oxidation activity. The reason behind this observation may be the presence of reactive oxygen species (ROS). The reactivity of pyrite during the exposure to an aqueous solution has been previously reported to result in the generation of ROS, such as hydrogen peroxide (H2O2), superoxide anion radicals ðO2 Þ and hydroxyl radicals ( OH). The chemical reaction is as follows (28): Py ðIIÞ þ O2 /FeðIIIÞ þ ðO2 Þ
(3)
Py II þ O$2 /Fe III þ H2 O2
(4)
Fe II þ H2 O2 /$OH þ OH þ Fe III
(5)
As the above reaction equation illustrates, the ROS concentration in the leaching solution would increase with the DO concentration. And it was well known that ROS was particularly toxic to cells (29,30). Therefore, the presence of ROS during biooxidation at higher DO concentrations may explain why the Fe2þ oxidation activity did not greatly increase along with the rus gene
1. Dew, D., Lawson, E., and Broadhurst, J.: The BIOXÒ process for biooxidation of gold-bearing ores or concentrates, pp. 45e80, in: Rawlings, D. E. (Ed.), Biomining: theory, microbes and industrial processes. Springer-Verlag, Berlin (1997). 2. Ingledew, W. J.: Thiobacillus ferrooxidans. The bioenergetics of an acidophilic chemolithotroph, Biochim. Biophys. Acta, 683, 89e117 (1982). 3. De Kock, S., Barnard, P., and Plessis, C.: Oxygen and carbon dioxide kinetic challenges for thermophilic mineral bioleaching processes, Biochem. Soc. Trans., 32, 273e275 (2004). 4. d’Hugues, P., Cezac, P., Cabral, T., Battaglia, F., Truong-Meyer, X. M., and Morin, D.: Bioleaching of a cobaltiferous pyrite: a continuous laboratory-scale study at high solids concentration, Miner. Eng., 10, 507e527 (1997). 5. Liu, M. S., Branion, R. M. R., and Duncan, D. W.: The effects of ferrous iron, dissolved oxygen, and inert solids concentrations on the growth of thiobacillus ferrooxidans, Can. J. Chem. Eng., 66, 445e451 (1988). 6. Gericke, M., Pinches, A., and van Rooyen, J. V.: Bioleaching of a chalcopyrite concentrate using an extremely thermophilic culture, Int. J. Miner. Process., 62, 243e255 (2001). 7. Guay, R., Silver, M., and Torma, A. E.: Ferrous iron oxidation and uranium extraction by Thiobacillus ferrooxidans, Biotechnol. Bioeng., 19, 727e740 (1977). 8. Gleisner, M., Herbert, J. R. B., and Frogner Kockum, P. C.: Pyrite oxidation by Acidithiobacillus ferrooxidans at various concentrations of dissolved oxygen, Chem. Geol., 225, 16e29 (2006). 9. Silverman, M. and Lundgren, D.: Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans: I. An improved medium and a harvesting procedure for securing high cell yields, J. Bacteriol., 77, 642e647 (1959). 10. Vogel, A. L.: A textbook of quantitative inorganic analysis. Longman, London (1961). 11. Davis, D. G. and Jacobsen, W. R.: Determination of iron and iron-aluminum mixtures by titration with EDTA, Anal. Chem., 32, 215e217 (1960). 12. Bandyopadhyay, B., Humphrey, A., and Taguchi, H.: Dynamic measurement of the volumetric oxygen transfer coefficient in fermentation systems, Biotechnol. Bioeng., 9, 533e544 (1967). 13. Sugio, T., Taha, T. M., Kanao, T., and Takeuchi, F.: Increase in Fe2þ-producing activity during growth of Acidithiobacillus ferrooxidans ATCC23270 on Sulfur, Biosci. Biotechnol. Biochem., 71, 2663e2669 (2007). 14. Saywell, L. and Cunningham, B.: Determination of iron: colorimetric o-phenanthroline method, Ind. Eng. Chem. Anal. Ed., 9, 67e69 (1937). 15. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 16. Schmittgen, T. D. and Livak, K. J.: Analyzing real-time PCR data by the comparative CT method, Nat. Protoc., 3, 1101e1108 (2008). 17. Liu, H. L., Chen, B. Y., Lan, Y. W., and Cheng, Y. C.: SEM and AFM images of pyrite surfaces after bioleaching by the indigenous Thiobacillus thiooxidans, Appl. Microbiol. Biotechnol., 62, 414e420 (2003). 18. Ndlovu, S. and Monhemius, A. J.: The influence of crystal orientation on the bacterial dissolution of pyrite, Hydrometallurgy, 78, 187e197 (2005). 19. Pisapia, C., Humbert, B., Chaussidon, M., and Mustin, C.: Perforative corrosion of pyrite enhanced by direct attachment of Acidithiobacillus ferrooxidans, Geomicrobiol. J., 25, 261e273 (2008). 20. Rojas-Chapana, J. A. and Tributsch, H.: Interfacial activity and leaching patterns of Leptospirillum ferrooxidans on pyrite, FEMS Microbiol. Ecol., 47, 19e29 (2004). 21. Garcia-Ochoa, F., Gomez, E., Santos, V. E., and Merchuk, J. C.: Oxygen uptake rate in microbial processes: an overview, Biochem. Eng. J., 49, 289e307 (2010). 22. Deveci, H.: Effect of solids on viability of acidophilic bacteria, Miner. Eng., 15, 1181e1189 (2002). 23. Dumsday, G. J., Ocal, G., Bridger, J. S., and Zachariou, M.: The use of oxygen uptake rate to monitor discovery of microbial and enzymatic biocatalysts, Biotechnol. Bioeng., 102, 673e683 (2009). 24. Quatrini, R., Appiaayme, C., Denis, Y., Ratouchniak, J., Veloso, F., Valdes, J., Lefimil, C., Silver, S., Roberto, F., and Orellana, O.: Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling, Hydrometallurgy, 83, 263e272 (2006).
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25. Cox, J. and Boxer, D.: The purification and some properties of rusticyanin, a blue copper protein involved in iron (II) oxidation from Thiobacillus ferrooxidans, Biochem. J., 174, 497e502 (1978). 26. Yarzábal, A., Duquesne, K., and Bonnefoy, V.: Rusticyanin gene expression of Acidithiobacillus ferrooxidans ATCC 33020 in sulfur- and in ferrous iron media, Hydrometallurgy, 71, 107e114 (2003). 27. Liu, W., Lin, J., Pang, X., Cui, S., Mi, S., and Lin, J.: Overexpression of rusticyanin in Acidithiobacillus ferrooxidans ATCC 19859 increased Fe(II) oxidation activity, Curr. Microbiol., 62, 1e5 (2010).
J. BIOSCI. BIOENG., 28. Cohn, C. A., Laffers, R., Simon, S., O’Riordan, T., and Schoonen, M.: Role of pyrite in formation of hydroxyl radicals in coal: possible implications for human health, Part. Fibre Toxicol., 3, 1e10 (2006). 29. Imlay, J. A.: Cellular defenses against superoxide and hydrogen peroxide, Annu. Rev. Biochem., 77, 755e776 (2008). 30. Jones, G. C., Corin, K. C., van Hille, R. P., and Harrison, S. T. L.: The generation of toxic reactive oxygen species (ROS) from mechanically activated sulphide concentrates and its effect on thermophilic bioleaching, Miner. Eng., 24, 1198e1208 (2011).
Effects of dissolved oxygen on the biooxidation process of refractory gold ores Li-Xin Sun, Xu Zhang,* Wen-Song Tan, and Ming-Long Zhu The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China Received 18 April 2012; accepted 7 June 2012 Available online 9 August 2012
While multiple theories exist regarding the effect of dissolved oxygen (DO) on the biooxidation of minerals, few studies have been performed the cellular or molecular scale (e.g., genetics) and the mechanism remains unclear. In this paper, the effects of DO concentration on the biooxidation process of refractory sulfide gold ores by Acidithiobacillus ferrooxidans were investigated in the experimental stirred tank bioreactors (STRs). The results indicated that higher biooxidation and cell growth rates were correlated with higher DO concentration. The biooxidation process was restricted at 1.2 ppm DO due to oxygen limitation. Furthermore, the effects of DO on cellular and molecular scale were studied for the first time. The results demonstrated that the oxygen uptake rate (OUR), the Fe2D oxidation activity and the rus gene expression of A. ferrooxidans all increased with the DO concentration, which might be responsible for the increase of the biooxidation rates with the DO concentration. This study provides insight into the potential impact of molecular-level mechanisms of DO in the biooxidation process of minerals. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Refractory sulfide gold ores; Biooxidation process; Dissolved oxygen; Acidithiobacillus ferrooxidans; Stirred tank bioreactors]
Biooxidation of sulfidic-refractory gold minerals is often applied in the pretreatment of refractory sulfide gold ores. Currently, the commercial process of the biooxidation had been successfully used in the gold processing (1). Acidithiobacillus ferrooxidans, one of the primary organisms in the commercial biooxidation industries, is an acidophilic chemolithotrophic gram-negative bacterium. A. ferrooxidans obtains energy by the oxidation of ferrous ions to ferric ions and uses molecular oxygen as the terminal electron acceptor in aerobic conditions (2). Therefore, the dissolved oxygen (DO) plays an important role in the biooxidation process of refractory gold ores. Previous reports had indicated that a minimum concentration of DO (1.2e1.5 ppm) was required to avoid the limitation (3,4) (Dew, D. W and Miller, G., Abstr. IBS97, Aus., p. M7.1.1-M7.1.9, 1997). Additionally, Liu et al. (5) found that A. ferrooxidans did not grow in 9K medium, if the DO concentration was below 0.20 mg/L. Gericke et al. (6) claimed that the DO concentration limitation was 50% during the bioleaching of chalcopyrite concentrate using an extremely thermophilic culture. Furthermore, it had been found that both the cell specific growth rate and oxidation rate increased with the oxygen concentration (7,8). In order to ensure the DO concentration is maintained above limitation concentration, the oxygen mass transfer rate was often heightened by increasing either aeration or agitation intensity. However, these options would result in additional energy
consumption. Conversely, they may inhibit the microbial growth and biooxidation rates if, for example, the agitation intensity is beyond a certain level (4). To date, few studies have been performed on the effects of DO at a cellular or molecular scale (e.g., genetics), which limited the understanding of the mechanism and the potential optimization methods for the biooxidation process. To address this need, in this work, the effects of DO on the biooxidation rates, the activity of bacterial cells, the Fe2þ oxidation activity and the expression levels of rus genes of A. ferrooxidans were investigated in the experimental STRs. The results of this study were expected to further the current understanding of the effects of the DO concentration on the biooxidation process and provide scientific guidance for the design and optimization of the biooxidation process.
* Corresponding author at: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, P. O. Box 309, Shanghai 200237, PR China. Tel.: þ86 21 64252536; fax: þ86 21 64252250. E-mail address: [email protected] (X. Zhang).
Experimental procedures The batch biooxidation experiments were carried out in three 1.5 L STRs with 900 ml of iron-free 9K medium, 100 ml of the inoculated bacterial culture and 100 g of ores as energy source. The agitation was provided by a six-bladed 45 downward pitched blade turbine (PBT) impeller. A sparger was
MATERIALS AND METHODS Sample The low-grade refractory gold ores used in the experiments were kindly supplied by Tiancheng Co., Ltd., Shandong, P. R. China, containing 24.9 g/t Au, 96.1 g/t Ag, 21.30% Fe, 20.09% S, 7.74% C, 4.59% As, 0.02% Cu, 0.31% Pb and 0.13% Zn. Xray diffraction (XRD) analysis showed that the major components of the sample was pyrite and quartz. Microorganism and culture medium The strain of A. provided by Tiancheng Co., Ltd. The culture medium (iron-free developed from the 9K medium (9). The composition was (NH4)2SO4, 3.0; KCl, 0.1; K2HPO4, 0.5; MgSO4$7H2O, 0.5; Ca chemical reagents were of analytical grade.
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.06.004
ferrooxidans was 9K medium) was as follows (g/L): (NO3)2, 0.01. All
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situated below the bottom impeller. The dry air aeration rate was measured by a gas rotameter and controlled by a valve attached to the rotameter. The aeration rate was adjusted from 0.02 to 2 L/min, to stabilize the DO concentration at 5.2 ppm, 3.1 ppm or 1.2 ppm (20%, 50% and 80% of the saturation value, respectively) during the biooxidation process. The environmental conditions were as follows: 41 C, pH 1.5, the impeller speed in the STRs was 300 rpm. Analytical methods The pH and the DO concentration of the suspension were measured by a pH meter (Mettler model FE20) and a DO meter (OXYFERM 225, Hamilton). The mineral samples were collected after biooxidation for SEM (JEOL JSM-6360 LV) analysis. Mineral dissolution was determined by measuring the iron concentration in solution. The concentration of ferrous iron was determined by titration with potassium dichromate in the presence of the indicator N-phenylanthranilic acid (10). The ferric iron concentration was determined by titration EDTA at pH 2 in the presence of the indicator sulphosalicylic acid (11). Total iron concentration in the liquid phase was the summation of the ferric and ferrous iron concentrations. The OUR was measured using the dynamic gas-out/gas-in method (12). The number of free cells in solution was measured by direct microscopic counting using a Pettrof-Hauser-type cell counter. The ores oxidation ratio (l) and oxidation rate (y) were calculated as follows (8):
l ¼
y ¼
½Feti ½Feto ½Fep ½Fetj ½Feti tj ti
! 100%
(1)
(2)
where ½Feti and ½Fetj are the iron concentrations in the liquid at two consecutive time points, respectively. ½Fet0 and [Fe]p are the iron concentrations in the liquid and in the ores, respectively, prior to biooxidation. All experiments were performed in triplicate to reduce the likelihood that our findings were taking place by chance. The Fe2þ oxidation activity of A. ferrooxidans at Fe2D oxidation activity different DO concentrations was determined by measuring the concentration of oxidized Fe2þ in the reaction mixture under aerobic conditions (13). The reaction mixture was composed of 2 ml 0.1 M alanine-SO24 buffer (pH 3.0), washed intact bacterial cells, and 1 ml ferrous sulphate (CFe2þ ¼ 0:2 g=L). The total volume of the mixture was 3.0 ml. The reaction was carried out by shaking the reaction mixture at 41 C. A sample of the reaction mixture (0.2 ml) was withdrawn every 15 s. The concentration of Fe2þ was then determined spectrophotometrically using the o-phenanthroline method (14). Protein concentration in the mixture was measured with the Bradford method (15), using crystalline bovine serum albumin as the reference protein. The Fe2þ oxidation activity was then defined as the decrease of ferrous ion concentration per second per gram of dry cell mass.
Quantitative real-time PCR The relative expression levels of the rus gene of A. ferrooxidans grew at different DO concentrations was determined with real-time PCR, using the Mx3000 Real-Time PCR System (Stratagene, La Jolla, CA, USA). The cell were collected during the exponential phase by filtration of the liquid through a 0.22 mm pore-size membrane. Then, the total cellular RNA was extracted using the TRIzol reagent (TianGen Biotech, Beijing, P.R. China) according to the manufacturer’s instructions. The synthesis of cDNA from RNA samples was performed using the 1st Strand cDNA synthesis kit (MBI, USA), according to the manufacturer’s instructions. These reaction mixtures were incubated at 50 C for 30 min, and the reaction was terminated by heating the samples at 85 C for 5 min. Quantitative RT-PCRs of the resultant cDNA were performed with the Mx3000 Real-Time PCR System and the Maxima SYBR Green q-PCR Master Mix Kit (MBI, America). The reaction mixture contained 12.5 ml of SYBRÒ Green PCR Master Mix (Biotools), 1 mL of cDNA, 1 mL of the corresponding, and 0.5 ml of each primer (detailed below). The mixture was brought 25 ml of total volume with nuclease-free water. The amplification program consisted of 1 cycle at 95 C for 5 min, and then 40 cycles at 95 C for 30 s, 58 C for 60 s and 72 C for 60 s. The real-time PCR primers were 50 -CGCGATGGCCGGTACTCTGGA-30 and 50 -CCGCCGCGACCACATGTACAGT-30 for the rus gene, 5-AATCCAAGAAGAAGCACCG-30 and 50 -CCACTGATGTTCCTCCAG-30 for the 16S rRNA (a house-keeping gene, as a control). The threshold cycle (Ct) values were determined using the curve analytical software of the Mx3000 system (Stratagene). The fold change in the target gene was determined by the formula 2DDCt (16), where DDCt ¼ (DCt from the experimental condition) (DCt from control). The DCt was calculated as the difference between the Ct of the target gene and Ct of the reference gene (16S rRNA).
RESULTS AND DISCUSSION Effects of DO on the mineral oxidation The concentration of Fe2þ and Fe3þ in solution, the ores oxidation ratio and the biooxidation rate during the biooxidation process at various DO concentrations are shown in Fig. 1. As shown in Fig. 1AeC, the concentration of Fe3þ and the oxidation ratio at 1.2 ppm DO were lower than those at 5.2 ppm DO. However, the concentration of Fe2þ was higher at 1.2 ppm DO than at 5.2 ppm DO. It should be noted that the biooxidation process was inhibited at 1.2 ppm DO due to oxygen limitation. The concentration of oxygen limitation was closed to 1.5 ppm which was proposed by Dew and Miller (Dew, D. W. and Miller, G., Abstr. IBS97, Aus., p. M7.1.1-M7.1.9, 1997).
FIG. 1. Evolutions of the concentration of Fe2þ (A) and Fe3þ (B), the ores oxidation ratio (C) and rate (D) at various DO concentrations.
VOL. 114, 2012 It illustrated from Fig. 1D that the oxidation rate increased with the growth process during the first 160 h and then decreased. Conversely, the oxidation rate increased with the DO concentration. The highest observed biooxidation rate was about 0.087 g/L/h at 5.2 ppm DO, but decreased to 0.057 g/L/h at 1.2 ppm DO. The biooxidation rate at 5.2 ppm was only slightly higher than the rate at 3.1 ppm. As the terminal electron acceptor, the availability of the DO was observed to be the limiting factor during Fe2þ oxidation by A. ferrooxidans. Therefore, adequate DO availability could accelerate the process of Fe2þ oxidization to Fe3þ. These results agreed well with the found of Gleisner et al. (8). The SEM micrographs of the oxidized mineral are shown in Fig. 2AeE. These micrographs illustrate that while the mineral surface was smooth prior to the biooxidation process (Fig. 2A), but became rough after the biooxidation process (Fig. 2BeD). As displayed in Fig. 2B and C, there were obviously several holes of approximately 1e2 mm in length on the mineral surface. These holes were not observed under abiotic conditions. Instead, irregular long and narrow cracks were observed on the mineral surface before biooxidation (Fig. 2E). A similar observation had been reported in previous studies (17e20). It was hypothesized that the
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holes on the mineral surface were caused by the adhesion of bacterial cells, and the cracks developed from initial mineral defects. These corrosion pits were all conducive to the mineral oxidation. Effects of DO on the suspended cells growth and the activity of bacterial The effect of DO concentration on the growth of suspended bacterial cells during the biooxidation process is shown in Fig. 3. At 5.2 ppm DO, the cells grew markedly during the exponential growth phase without a lag phase, and the growth process reached the stationary phase after 138 h. However, at 3.1 ppm and 1.2 ppm DO, the concentration of suspended cells decreased during the first 24 h due to the adhesion of the cells onto the mineral surface. The exponential growth periods were 156 h at 3.1 ppm DO and 186 h at 1.2 ppm DO. These results showed that the exponential growth phase of A. ferrooxidans was shorter at higher DO concentrations than at lower DO concentrations. Thus, it could hypothesize that the cells’s growth was restrained at lower DO concentration. However, eventually all three experimental groups reached the same final concentrations, approximately 1.6 108 cell/ml. It was different from the results of Gleisner et al. (8) that the concentration of
FIG. 2. The SEM micrographs of mineral surface before (A) and after biooxidated by A. ferrooxidans at 5.2 ppm DO (B), 3.1 ppm DO (C), 1.2 ppm DO (D) and abiotic (E) as control.
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FIG. 5. The Fe2þ oxidation activity of A. ferrooxidans during the exponential growth phase at various DO concentrations. FIG. 3. Evolutions of the suspended cell concentration at various DO concentrations.
A. ferrooxidans generally increased with the DO concentration. However, in that study, the final concentration of cells was 1e20 106 cell/ml, which was one order of magnitude smaller in number that those used in this study. Thus, the difference in the cell concentration might be responsible for differences in the results. The oxygen uptake rate (OUR) which could provide important information about the metabolic activity of the cultured cells, has been previously used for optimizing the fermentation process (21). The OUR of bacteria is based on the presumption that only the active cells will oxidize ferrous iron and hence consume oxygen (22). Fig. 4 shows the relationship between DO concentration and the OUR of A. ferrooxidans during the biooxidation process. It could be seen that OUR increased during the bacterial exponential growth phase, reached the maximum in the late period of cell growth and subsequently decreased. Because the Fe2þ oxidization rate increased as the cells grew in the exponential growth phase, the OUR also initially increased. Then, as the cells began to die and the overall metabolic activity decreased, the OUR also decreased. In addition, the OUR was greater at higher DO concentrations. The maximum OUR was 0.052 mg/L/s at 5.2 ppm DO, with a sharp decrease to 0.034 mg/L/s at 1.2 ppm DO. This finding indicated that the activity of the bacterial population increased with the DO concentration.
FIG. 4. Evolution of the OUR during the biooxidation at various DO concentrations.
Effect of DO on the Fe2D oxidation activity of A. ferrooxidans There is a positive correlation between the biomass concentration and the observed OUR, which is intrinsically linked in the activity of bacterial population (23). However, the OUR could not directly reflect the oxidation activity of any individual cell. The Fe2þ oxidation activity of A. ferrooxidans during the exponential growth phase was determined in this paper and is shown in Fig. 5. The results indicated that the Fe2þ oxidation activity increased with the DO concentration, with activity observed to be w 25% higher at 5.2 ppm DO than that at 1.2 ppm DO. In aerobic conditions, A. ferrooxidans obtains energy from the oxidation process of ferrous ions to ferric ions using molecular oxygen as the terminal electron acceptor in the respiratory chain (2). Additionally, the cells also get reducing power derived from the respiratory chain. Both of them are necessary for many subsequent metabolic processes, including CO2 fixation (24). The Fe2þ oxidation activity of A. ferrooxidans could reflect both the electron transport rate of respiratory chain and the oxidation activity of the individual cell. The higher Fe2þ oxidation activity, the higher electron transport rate of respiratory chain. Namely, bacteria may get more energy and reducing power at higher DO concentration, resulting in an increase in the metabolic activity of the cells. Effect of DO on expression of rus genes of A. ferrooxidans Several redox proteins involved in the electron transfer process have been isolated and characterized in A. ferrooxidans. Among these, the 16.5-kDa periplasmic copper protein rusticyanin (encoded by the rus gene), have received special attention because it may constitute as much as 5% of the total soluble proteins in A. ferrooxidans during autotrophic growth on iron (25). It had been suggested that rusticyanin functioned as an electron reservoir by readily took up electrons available at the outer membrane and then channelled them through the respiratory pathway. The study of Yarzábal et al. (26) showed that the iron oxidation took place only when the rusticyanin had reached a critical intracellular level. Liu et al. (27) also found that by overexpressing the rus gene, it was possible to increase the Fe2þ oxidation activity. In order to study the effect of DO concentration on the Fe2þ oxidation activity, the effect of DO concentration on the rus gene expression of A. ferrooxidans was determined. The relative transcription levels of the rus gene at the three DO concentrations are shown in Fig. 6. It was demonstrated that the expression levels of the rus gene of A. ferrooxidans also increased with DO concentration. The rus gene
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expression. However, future studies will be required to confirm this hypothesis. ACKNOWLEDGMENTS This study was financially supported by Open Project Funding of State Key Laboratory of Bioreactor Engineering of China and the National High Technology Research and Development Program of China (Nos. 2007AA060904 and 2012AA061503). References
FIG. 6. Effect of DO concentration on the expression of the rus gene during the biooxidation of A. ferrooxidans (rus gene expression in 1.2 ppm DO condition was designated as control).
expression was restrained at 1.2 ppm DO, which might lead to a lack of intracellular rusticyanin in A. ferrooxidans. This lack of rusticyanin might have ultimately resulted in the observed decrease of the Fe2þ oxidation activity and the restriction of the biooxidation process at 1.2 ppm DO. It could be found from Figs. 5 and 6, respectively, that the rus gene of A. ferrooxidans increased 60% and the Fe2þ oxidation activity increased 22%, when the DO concentration increased from 1.2 ppm to 3.1 ppm. Thus, increased the rus gene expression was an effective way to improve the Fe2 þ oxidation activity. It can be hypothesized that, if the rus gene expression of A. ferrooxidans could be upregulated artificially at the lower DO concentration, the Fe2þ oxidation activity cells would increase, thereby accelerating the rate of ore biooxidation. The research of Liu et al. (27) provided such an approach by overexpressing recombinant rus gene products in an engineered strain of A. ferrooxidans (pTRUS) containing a homologous rus gene. However, this method could achieve the desired goals at lower DO concentrations. Future studies will need to be conducted to test if this is the case. However, the expression levels of the rus gene increased by 130%, but Fe2 þ oxidation activity increased by only 4%, when the DO concentration increased from 3.1 ppm to 5.2 ppm. Therefore, it appeared that the increase in DO concentration could not improve the Fe2þ oxidation activity. The reason behind this observation may be the presence of reactive oxygen species (ROS). The reactivity of pyrite during the exposure to an aqueous solution has been previously reported to result in the generation of ROS, such as hydrogen peroxide (H2O2), superoxide anion radicals ðO2 Þ and hydroxyl radicals ( OH). The chemical reaction is as follows (28): Py ðIIÞ þ O2 /FeðIIIÞ þ ðO2 Þ
(3)
Py II þ O$2 /Fe III þ H2 O2
(4)
Fe II þ H2 O2 /$OH þ OH þ Fe III
(5)
As the above reaction equation illustrates, the ROS concentration in the leaching solution would increase with the DO concentration. And it was well known that ROS was particularly toxic to cells (29,30). Therefore, the presence of ROS during biooxidation at higher DO concentrations may explain why the Fe2þ oxidation activity did not greatly increase along with the rus gene
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