The aim of this study was to resolve the apparent inconsistency between the prevalence of the moderate thermophile Sulfobacillus thermosulfidooxidans in copper sulfide heaps and the sensitivity of this bacterium to the solvent extraction (SX) reagent used to concentrate and purify the copper in solution prior to metal production. More than 1 year was required to achieve habituation to the most inhibitory component of the SX reagent, 4-nonylphenol (4-NP; 2 mg L-1), when S. thermosulfidooxidans was grown in ferrous ion medium. However, habituation to 4-NP did not confer the ability to tolerate higher 4-NP concentrations and those characteristics acquired by the cells during habituation were rapidly lost when grown for a short period in 4-NP-free medium. The culture habituated to the presence of 2 mg L-1 4-NP exhibited differences in cell morphology, cell ultrastructure and membrane fatty acid composition compared with the untreated culture subjected to a similar subculture regime. It is concluded that, where S. thermosulfidooxidans is prevalent in copper sulfide heaps associated with SX-EW plants, this species has become habituated through continuous long-term exposure to low concentrations of 4-NP derived from entrained SX reagent in the recycled leach solutions. (C) 2010 Elsevier Ltd. All rights reserved.