Document Type : Full article


1 Biotechnology Research Laboratory, School of Chemical, Petroleum and Gas Engineering, Iran University of Science and Technology, P. O. Box: 16846-13114, Tehran, Iran

2 Biotechnology Research Laboratory, School of Chemical, Petroleum and Gas Engineering, Iran University of Science and Technology, P.O. Box: 16846-13114, Tehran, Iran


In this study, the effects of some factors on bacterial growth and ferrous oxidation rates were investigated by Acidithiobacillus ferrooxidan in 250 ml shake flasks. One factor at a time (OFAT) design approach was used for preliminary evaluation of various factors affecting the process, such as pH, initial ferrous and elemental sulfur concentrations, shaker agitation rate, and liquid to flask volume ratio. After that, optimal levels of effective last three factors to achieve high oxidation rate and cell growth rate were investigated using a full factorial design. It was obtained that agitation rate and liquid to flask volume, as well as their binary interaction, are significant factors on ferrous iron bio-oxidation rate. In contrast, initial high ferrous iron concentration was the only effective factor on the cell growth rate. Maximum bio-oxidation rate of 0.417 g/L was achieved at the media with Fe2+ ion concentration of 30 g/l, agitation rate of 200 rpm, and liquid to flask volume ratio of 20% by full factorial optimization, which is an about 40% increase compared to the result obtained in OFAT method. Then, the effect of step-wise adaptation of A. ferrooxidans to in high Fe2+ concentration was studied, and about 40% reduction in bacterial lag phase time, and 36 and 86% increase in bacterial growth rate and bio-oxidation rate were acquired, respectively.


Main Subjects

[1]      Kahrizi, E., Alemzadeh, I. and Vossoughi, M., “Bio-oxidation of ferrous ions by Acidithiobacillus ferrooxidans in a monolithic bioreactor”, J. Chem. Tech. Biotech., 84 (4), 504 (2009).
[2]      Mousavi, S. M., “Influence of process variables on biooxidation of ferrous sulfate by an indigenous Acidithiobacillus ferrooxidans, Part I: Flask experiments”, Fuel, 85 (17-18), 2555 (2006).
[3]      Mousavi, S. M., Yaghmaei, S. and Jafari, A., “Influence of process variables on biooxidation of ferrous sulfate by an indigenous Acidithiobacillus ferrooxidans, Part II: Bioreactor experiments”, Fuel, 86 (7-8), 993 (2007).
[4]      Mousavi, S. M., Yaghmaei, S., Jafari, A., Vossoughi, M. and Ghobadi, Z., “Optimization of ferrous biooxidation rate in a packed bed bioreactor using Taguchi approach”, Chem. Eng. Process: Process Intensification, 46 (10), 935 (2007).
[5]      Khavarpour, M., Najafpour, G. D., Ghoreyshi, A. A., Jahanshahi, M. and Bambai, B., “Enhanced Fe2+ oxidation by mixed culture originated from hot spring: Application of response surface method”, Afr. J. Biotechnol., 10 (19), 3769 (2011).
[6]      Mesa, M. M., Andrades, J. A., Macıas, M. and Cantero, D., “Biological oxidation of ferrous iron: study of bioreactor efficiency”, J. Chem. Technol. Biotechnol., 79 (2), 163 (2004).
[7]      Cabrera, G., Gómez, J. M. and Cantero, D., “Influence of heavy metals on growth and ferrous sulphate oxidation by Acidithiobacillus ferrooxidans in pure and mixed cultures”, Process Biochem., 40 (8), 2683 (2005).
[8]      Kupka, D., Rzhepishevska, O. I., Dopson, M., Lindström, E. B., Karnachuk, O. V. and Tuovinen, O. H., “Bacterial oxidation of ferrous iron at low temperatures”, Biotechnol. Bioeng., 97 (6), 1470 (2007).
[9]      Malhotra, S., Tankhiwale, A. S., Rajvaidya, A. S. and Pandey, R. A., “Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans”, Bioresource Technol., 85 (3), 225 (2002).
[10]  Barron, J. L., Lueking, D. R., “Growth and maintenance of Thiobacillus ferrooxidans cells”. Appl. Environ. Microbiol., 56 (9), 2801 (1990).
[11]  Gomez, J. M. and Cantero, M., “Kinetic study of biological ferrous sulphate oxidation by iron-oxidising bacteria in continuous stirred tank and packed bed bioreactors”, Process Biochem., 38 (6), 867 (2003).
[12]  Wu, X., Zhang, Z., Dend, F. and Liu, X., “Molecular characterization of Acidithiobacillus ferrooxidans strains isolated from different environments by three PCR-based methods”, Journal of Central South University, 22 (2), 455 (2015).
[13]  Savić, D. S., Veljković, V. B., Lazić, M. L., Vrvić, M. M. and Vučetić, J. I., “Effects of the oxygen transfer rate on ferrous iron oxidation by Thiobacillus ferrooxidans”, Enzyme Microb. Technol., 23 (7-8), 427 (1998).
[14]  Liu, Y. S., Wu, J. Y. and Ho, K. P., “Characterization of oxygen transfer conditions and their effects on Phaffia rhodozyma growth and carotenoid production in shake-flask cultures”, Biochem. Eng. J., 27 (3), 331 (2006).
[15]  Wahid, Z. and Nadir, N., “Improvement of one factor at a time through design of experiments”, World Applied Sciences J., 21 (1), 56 (2013).
[16]  Frey, D. D. and Wang, H., “Adaptive one-factor-at-a-time experimentation and expected value of improvement”, Technometrics, 48 (3), 418 (2006).
[17]  Montgomery, D. C., Design and analysis of experiments, 7th ed., John Wiley & Sons, Inc., New York, USA, (2009).
[18]  Eriksson, L., Johansson, E., Kettaneh-Wold, N., Wikstrom, C. and Wold, S., Design of experiments: Principles and application, MKS Umetrics AB, 3rd ed., Stockholm, Sweden, (2008).
[19]  Rahman, S. and Gagnon, G. A., “Bench-scale evaluation of ferrous iron oxidation kinetics in drinking water: Effect of corrosion control and dissolved organic matter”, J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng., 49 (1), 1 (2014).
[20]  Zhang, C., Min, X., Chai, L. and Zhong, H., “Influencing factors of lag phase in growth of Thiobacillus ferrooxidans”, J. Cent. South Univ. Technol., 30 (5), 489 (1999).
[21]  Nemati, M., Harrison, S. T. L., Hansford, G. S. and Webb, C., “Biological oxidation of ferrous sulphate by Thiobacillus ferrooxidans: A review on the kinetic aspects”, Biochem. Eng. J., 1 (3), 171 (1998).
[22]  Kim, D. J., Pradhan, D., Ahn, J. G. and Lee, S. W., ‘Enhancement of metals dissolution from spent refinery catalysts using adapted bacteria culture-effects of pH and Fe (II)”, Hydrometallurgy, 103 (1-4), 136 (2010).
[23]  Harvey, A. E., Smart, J. A. and Amis, E. S., “Simultaneous spectrophotometric determination of iron (II) and total iron with 1,10-phenanthroline”, Anal. Chem., 27 (1), 26 (1955).
[24]  Meruane, G. and Vargas, T., “Bacterial oxidation of ferrous iron by Acidithiobacillus ferrooxidans in the pH range 2.5-7.0”, Hydrometallurgy, 71 (1-2), 149 (2003).
[25]  Mukherjee, C., Jones F. S., Bigham J. M. and Tuovinen O. H., “Synthesis of argentojarosite with simulated bioleaching solutions produced by Acidithiobacillus ferrooxidans”, Mater. Sci. Eng.: C Mater. Biol. Appl., 66, 164 (2016).
[26]  Pradhan, D., Kim, D. J., Ahn, J. G., Roy Chaudhury, G. and Lee, S. W., “Kinetics and statistical behavior of metals dissolution from spent petroleum catalyst using acidophilic iron oxidizing bacteria”, J. Indust. Eng. Chem., 16 (5), 866 (2010).
[27]  Nazari, B., Jorjani, E., Hani, H., Manafi, Z. and Riahi, A. “Formation of jarosite and its effect on important ions for Acidithiobacillus ferrooxidans bacteria”, Transactions of Nonferrous Metals Society of China, 24 (4), 1152 (2014).
[28]  Sun, L. X., Zhang, X., Tan, W. S. and Zhu, M. L., “Effect of agitation intensity on the biooxidation process of refractory gold ores by Acidithiobacillus ferrooxidans”, Hydrometallurgy, 127-128, 99 (2012).
[29]  Sun, L. X., Zhang, X., Tan, W. S. and Zhu, M. L., “Effects of dissolved oxygen on the biooxidation process of refractory gold ores”, J. Biosci. Bioeng., 114 (5), 531 (2012).
[30]  Nemati, M. and Harrison, S. T. L., “Comparative study on thermophilic and mesophilic biooxidation of ferrous iron”, Miner. Eng., 13 (1), 19 (2000).
[31]  Abuhamed, T., Bayraktar, E., Mehmetoğlu, T. and Mehmetoğlu, Ü., “Kinetics model for growth of Pseudomonas putida F1 during benzene, toluene and phenol biodegradation”, Process Biochem., 39 (8), 983 (2004).
[32]  Rawlings, D. E., “Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates”, Microbial Cell Factories, 4 (13), 1 (2005).
[33]  Valdés, J., Pedroso, I., Quatrini, R., Dodson, R. J., Tettelin, H., Blake Ii, R., Eisen, J. A. and Holmes, D. S., “Acidithiobacillus ferrooxidans metabolism: From genome sequence to industrial applications”, BMC Genomics, 9 (597), 1 (2008).
[34]  Mousavi, S. M., Jafari, A., Yaghmaei, S., Vossoughi, M. and Turunen, I., “Experiments and CFD simulation of ferrous biooxidation in a bubble column bioreactor”, Comput. Chemical Eng., 32 (8), 1681 (2008).
[35]  Govender, E., Bryan, C. G. and Harrison , S. T. L., “Effect of physico-chemical and operating conditions on the growth and activity of Acidithiobacillus ferrooxidans in a simulated heap bioleaching environment”, Minerals Engineering, 75, 14 (2015).