Enhancing iron oxidation efficiency by a native strain of Acidithiobacillus ferrooxidans via response surface methodology, and characterization of proteins involved in metal resistance by proteomic approach

Document Type : Original Article


1 Islamic Azad University, Pharmaceutical Sciences Branch, Tehran, Iran

2 Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran

3 Faculty of Chemical Engineering, Sahand University of Technology, Tabriz, Iran


The effects of different factors on growth and bio-oxidation efficiency of a native strain of Acidithiobacillus ferrooxidans have been evaluated by the utilization of response surface methodology, RSM. Medium pH and iron concentration were found to be the most significant factors while temperature and ammonia concentrations had the least weight within the ranges investigated. Optimum operational conditions for maximizing Fe2+ oxidation were found to be 31 oC, 7 g/Liron concentration, 4.5 g/Ltotal ammonium salt concentration and medium pH 1.85. Maximum recovery of 98% of Zinc is the main outcome of results as observed at 7 g/Lof Fe2+, under optimized experimental conditions. The response of a bacterial strain to metals toxicity also studied.  The isolate showed good resistance to most of the toxic metals. The proteomics approach was used to identify the differentially expressed proteins under heavy metal stress. Four of the differentially expressed proteins were identified as major outer membrane protein of A. ferrooxidans, ribulose large bisphosphate carboxylase subunit, and holo synthase.


  1. Ehrlich, H.L., Past, present and future of biohydrometallurgy. Hydrometal, 2001, vol.59, pp. 127–134.
  2. Brierley, J.A., Brierley, C.L.,  Present and future commercial applications of biohydrometallurgy. Hydrometal, 2001, vol.59, pp. 233-239 (2001).
  3. Rao, K.S., Mishra, A., Pradhan, D., Chaudhary, G.R., Mohapatra, B.K.,  Das, T., Sukla, L.B., Mishra, B.K., Percolation bacterial leaching of low-grade chalcopyrite using acidophilic microorganisms. Korean J. Chem. Eng, 2008, vol. 25, pp. 524.
  4. Dew, D., Lawson, E., Broadhurst, J., The BIOX® process for biooxidation of gold-bearing ores or concentrates, Biomining: Theory, Microbes and Industrial Processes, D. Rawlings Eds., Springer Berlin Heidelberg, 1997.
  5. Deveci, H., Akcil, A., Alp, I., Materials Science and Technology 2003 Symposium: Process Control and Optimization in Ferrous and Non Ferrous Industry, Chicago, USA, 9-12 November, 2003.
  6. Wei, W., Zheng, Z., Liu, Y., Zhu, Z., J. Fermentation Bioeng, 1998, vol.26, pp. 395.
  7. Dutta, J.R., Dutta, P.K., Banerjee,R., Optimization of culture parameters for extracellularprotease production from a newly isolated Pseudomonas sp. using response surface and artificial neural networks.  Process Biochem, 2004, vol.39, pp. 2193-2198.
  8. Xiong, Y.H., Liu, J.Z., Song, H.Y., Ji, LN., Enhanced production of extracellular ribonuclease from Aspergillus niger by optimization of culture conditions using response surface methodology. Biochem. Eng. J, 2004, vol.21, pp.27-32.
  9. Kalil, S.J., Maugeri, F., Rodrigues, M.I., Response surface analysis and simulation as a tool for bioprocess design and optimization. Process Biochem, 2000, vol. 35, pp. 539-550.
  10.  Valenzuela, L., Chi, A., Beard, S., Orell, A., Guiliani, N., Shabanowitz, J., Hunt, D.F., Jerez, C.A., Biotechnol. Advances, 2006, vol.24, pp.197.
  11. Gygi, S.P., Corthals, G.L., Zhang, Y., Rochon, Y., Aebersold, R., Evaluation of two-dimensional electrophoresis-based proteome analysis. Proc Natl Acad Sci USA., 2000, vol.97, pp.9390-9395.
  12. Tuovinen, O.H., Kelly, D.P., Studies on the growth of Thiobacillus ferrooxidans Arch Microbiol, 1973, vol. 88, pp. 285-298 (1973)
  13. Leathen, W., Kinsel, N., Braley, I., Ferrobacillus ferrooxidans: a chemosynthetic autotrophic bacterium. J Bacteriol, 1956, vol. 72, pp. 700-704.
  14. Silverman, M.P., Lundgren, D.G.,Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans: I. An improved medium and a harvesting procedure for securing high cell yields. J Bacteriol, 1959, vol. 77, pp. 642-647
  15. Stark, A., Yankofsky, S., Regulation of amino acid transport in Thiobacillus thioparus.J Bacteriol, 1981, vol. 148, pp. 966-972.
  16. Karamanev, D.G., Nikolov, L.N., Mamatarkova, V., Rapid simultaneous quantitative determination of ferricand ferrous ions in drainage waters and similar solutions. Miner Eng, 2002, vol. 15, pp. 341-346.
  17. Draper, N.R., John, J.A., Response-surface designs for quantitative and qualitative variables. Technometrics, 1988, vol. 30, pp. 423-428.
  18. Myers, R.H., Montgomery, D.C., Response Surface Methodology. John Wiley & Sons Inc., New York, 2002.
  19. Murr, E.L., Torma, A.E., Brierley, J.A., Metallurgical Application of Bacterial Leaching and Related Microbiological Phenomena, Academic Press, New York, 1978, pp. 477-490.
  20. Nemati, M., Harrison, S., Hansford, G., Webb, C., Biological oxidation of ferrous sulphate by Thiobacillus ferrooxidans: a review on the kinetic aspects. Biochem Eng. J, 1998, vol. 1, pp. 171-190.
  21. Meruane, G., Vargas, T., Bacterial oxidation of ferrous iron by Acidithiobacillus ferrooxidans in the pH range 2.5–7.0. Hydrometal, 2003,vol. 71, pp. 149-158.
  22. Mousavi, S.M., Yaghmaei, S., Salimi, F., Jafari, A.,  Influence of process variables on biooxidation of ferrous sulfate by an indigenous Acidithiobacillus ferrooxidans. Part I: Flask experiments. Fuel, 2006, vol. 85, pp. 2555-2560.
  23. Smith, J.R., Luthy, G.R., Middleton, A.C., J Water Pollut Control Fed, 1988, vol. 60, pp. 518.
  24. Torma, A.E.,  The role of Thiobacillus ferrooxidans in hydrometallurgical processes, in Advances in Biochemical Engineering, 6, T.K. Ghosh, A. Fiechter and N. Blakebrough Eds., Springer-Verlag, Berlin-Heidelberg-New York, 1977.
  25. Gomez, C., Blazquez, M.L., Ballester, A.,Bioleaching of a Spanish Complex Sulphide Ore Bulk Concentrate. Miner Eng, 1999, vol, 12, pp.93-106.
  26. Okereke, A., Stevens, S.E., Kinetics iron oxidation by Thiobacilluse ferrooxidant. Appl Environ Microbiol, vol, 57, pp. 1052-1056 (1991).