IDentificación De A. Ferrooxidans y L. Ferrooxidans

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Hydrometallurgy 92 (2008) 102–106

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Hydrometallurgy
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t

Rapid and specific detection of Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans by PCR
Blanca Escobar ⁎, Karina Bustos, Gabriela Morales, Oriana Salazar
Centro de Hidro/Electrometalurgia, Departamento de Ingeniería Química y Biotecnología,Universidad de Chile, Beauchef 861, Santiago, Chile

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Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans are two of the most important bacteria in heap bioleaching processes of copper sulphide at common operating temperatures (18–24 °C). In this paper, both microorganisms were detected in solutions and ores coming from processes in 2 days, usingspecific amplification of 16S rDNA sequences by PCR. The technique was first validated using template DNA from pure cultures of the microorganisms. Then it was applied to samples of solutions and ores from bioleaching processes. Results were confirmed using tRFLP (terminal Restriction Fragment Length Polymorphism) with universal primers and by identification of isolated bacteria by means of culture. Thismethodology is more rapid and specific than the identification by tRFLP or by culture, which require from 1 to three weeks to positively detect the bacteria. The detection limit of this technique is 105 cells per ml. © 2008 Elsevier B.V. All rights reserved.

Article history: Received 7 December 2006 Received in revised form 4 December 2007 Accepted 31 January 2008 Available online 6 March 2008Keywords: Bioleaching A. ferrooxidans L. ferrooxidans PCR 16S rDNA

1. Introduction Copper sulphide ore bioleaching processes are currently under full development in Chile and worldwide, for treatment of secondary sulphide in heaps (Brierley, 1999), low-grade ores in dumps or for bioleaching of concentrates in reactors at high temperatures (ACL Project, 2000). In these processes, microorganisms(bacteria and archaea) play a major role as catalysts of chemical reactions, which in their absence, would be carried out at low speed, making the process inefficient (Norris, 1991). Amongst the microorganisms participating in these processes are iron-oxidising and sulphur-oxidising bacteria and archaea, heterotrophic bacteria, as well as other species that are active at different temperatures (Goebeland Stakebrandt, 1994; Yahya et al., 1999; Rawlings, 2001). Participation of these microorganisms in the dissolution of metal sulphides has been widely studied (Rohwerder et al., 2003). Today, it is accepted that iron-oxidising microorganisms are critical for keeping a high oxidising potential in solutions, which is a key condition for the dissolution of metal sulphides (Rawlings, 1998). Amongstmesophilic bacteria, the most important in bioleaching due to their oxidising capacity of Fe(II) to Fe(III) are Thiobacillus ferrooxidans and Leptospirillum ferrooxidans (Rawlings, 1998). T. ferrooxidans which was renamed Acidithiobacillus ferrooxidans by Kelly and Woods (2000), oxidises Fe(II) and reduced sulphur com-

⁎ Corrresponding author. E-mail address: bescobar@cec.uchile.cl (B....