Municipal solid waste (MSW) incineration fly ash used in this study was obtained from Tuas South Incineration Plant in Singapore. The fly ash was autoclaved at 121 °C for 15 min prior to use. A. niger was obtained from Dr H. Brandl (University of Zürich, Switzerland) and was cultured as previously described EPZ5676 solubility dmso [32]. 7-day old conidia were harvested from the surface of potato dextrose agar (Becton Dickinson Co.) using sterile deionized (DI) water. The number of spores was counted under a microscope (Olympus CX40) at 400× magnification using a Superior Marienfeld 0.1 mm depth haemocytometer. The spore suspension was diluted with DI water to

the desired spore suspension concentration (107 spores/ml). 1 ml of spore suspension was added to 100 ml of standard sucrose medium with composition (g/l): sucrose (100), NaNO3 (1.5), KH2PO4 (0.5), MgSO4∙7H2O (0.025), KCl (0.025), yeast extract (1.6), and incubated at 30 °C with rotary shaking at 120 rpm [32]. All reagents were of analytical grade. The liquid medium was autoclaved at 121 °C for 15 min prior to inoculation. Trichostatin A solubility dmso One-step bioleaching

was conducted following reported protocol [32]. In one-step bioleaching, the fungus was incubated with ash at 1% pulp density. Sterile medium was added to autoclaved flasks containing the fly ash, followed by inoculation of fungal spore suspension. Samples of fungi pellet were withdrawn after Day 7, 8, 17, and 27 for SEM, EDX and XRD analyses. In two-step bioleaching, the fungus was first cultured in an autoclaved sucrose medium (as in pure culture) and incubated at 30 °C with rotary shaking at 120 rpm

without fly ash. After 2 days, when a large pH drop occurred, sterile fly ash at 1% pulp density was added to the culture and the incubation was continued. Samples of fungi pellet were withdrawn after Day 2, 3, 7, 8, 17 and 27 for SEM, EDX and XRD analyses. Fungi pellet taken from pure culture, one-step bioleaching, and two-step bioleaching were washed with deionized water for three changes. The pellets were fixed with 3% (v/v) glutaraldehyde in deionized water at 4 °C overnight before being washed with deionized water Ribonucleotide reductase and dehydrated over an ethanol gradient. Samples were dried using a critical point dryer, mounted on copper stub and sputter-coated for 120 s using a JEOL JFC-1300 Auto Fine Coater fitted with a Pt target. A JEOL JSM-5600LV scanning electron microscope (SEM) was used to examine the morphology of the fungi and fly ash. For high magnifications, field emission scanning electron microscope (FESEM), JEOL JSM-6700F was used. The images obtained were analyzed using Image-Pro Premier software to obtain the size of particles and fungal hyphae. Energy-dispersive X-ray spectroscopy (EDX) (OXFORD Instruments 6647) was coupled to the SEM for surface elemental analysis of the fungal samples. The EDX data were analyzed using INCA Suite Version 4.01.