2.

Characterization of the ore samples

The pH samples were measured using pH meter. The elemental composition of the ores was determined by ED-XRF (Energy-Dispersive-X-Ray-Fluorescence) analysis. The mineral composition was determined by XRD (X-Ray Diffraction) analysis. The massive samples were prepared by inclusion of small piece of mineral between 0.8 and 1.0 cm diameter for the observations by ED-XRF and XRD. In addition, powder samples were dried at free atmosphere to preserve the surface for XRD analysis.

2.3

Microorganisms

A mixed culture of mixotrophic bacterial consortia (Comamonas testosteroni, Pantoea septica and Alicyclobacillus ferooxidans) used in the experiments was provided by isolation prior to this study. Previous studies by Giaveno and Donati (2001) have shown that mixed cultures are more efficient than pure cultures because of the cooperation of the microorganisms involved in the mixed cultures.

2.4

Bioleaching Experiment

Bioleaching experiments were performed in stirred pulp reactors, consisting of 500 mL and 250 mL glass flasks. The flask contained initially 300 mL mL of culture medium and 30 g mineral samples to obtain a pulp density of 10%wt. All isolates were mixed prior to bioleaching with the ratio per bacteria 1 : 1. Enrichment cultures were incubated for 1 to 2 weeks until they reached the optical density of 1 by using spectrophotometer and a chemical change occurred in the medium compared with an uninoculated control. The cultures were then incubated at room temperature and shaken at 250 rpm for up to 28 days. Leachate was removed at weekly intervals for determinations of soluble iron concentrations, total sulphate, pH and FDA (fluorescein diacetate) analysis. Concentration of total iron in leaching medium was determined by double beam atomic absorption spectrophotometer (AAS). Total sulphur and soluble sulphate concentration were determined using gravimetric method. All samples were prepared in triplicate.

2.5

Assay for FDA hydrolytic activity

Assay of fluorescein diacetate [3,6-diacethylfluorescein (FDA), Sigma-Aldrich Chemical Co., Milwaukee, WI] hydrolytic activity was conducted according to Green et al. (2006). Briefly, 1.5 g of air-dried sample were incubated with 50 ml of PBS buffer (pH 7.4) and 0.5 ml of 60 mM FDA substrate solution (20 mg in 10 ml acetone) in a water bath for 20 minute at 37 oC. The hydrolysis was terminated by the addition of 2 ml of iced acetone. The mixture was centrifuged (Beckman J2-HS) at 8000 rpm for 5 min and supernatants were transferred to cuvette. Absorbance was measured by using a spectrophotometer at a wavelength of 492 nm. Controls were performed by using 0.5 mL acetone instead of the fluorescein diacetate substrate solution. Concentration of fluorescein released was calculated by reference to a standard curve. The FDA analysis of all samples was carried out in triplicate.

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