5 on Day 7

during one-step bioleaching, and calcium oxalate precipitation occurs (Table 2). Indeed, the precipitation of calcium oxalate click here by several mycorrhizal species have also been documented, with one ascribed function being detoxification of calcium, since it is known that high concentration of free Ca2+ within cells is toxic [14], [15], [20] and [21]. Such a detoxification mechanism may also be the reason for the observed calcium oxalate precipitation in this present study. Interestingly, EDX data (Fig. 3b and e) and XRD data (Fig. 3f and Fig. 4a) show no evidence of precipitation of oxalates of aluminium, copper, iron, manganese, lead, and zinc during bioleaching, although others have reported fungal precipitation of oxalates and citrates of cobalt, copper, chromium, and nickel [12], [13], [22], [28] and [29]. This is expected since this website the concentration of these metal ions (at 101 ppm) in the fly ash is lower than that of calcium (103 ppm) in the liquid medium. Similar results were observed in two-step bioleaching. Samples were taken immediately after the addition of fly ash in two-step bioleaching. SEM photomicrographs confirmed the absence of solid particles on the fungal surface and within the section of the fungi pellet (data not shown). EDX results confirmed the presence of only carbon and oxygen; no metal element was detected within or outside the fungi pellet (data not shown). This

confirmed the absence of metal salt precipitation at the start of the two-step bioleaching. Fig. 4b shows the section of fungal pellet at

Day 7. The SEM photomicrograph at Day 7 (Fig. 4b) in two-step bioleaching was similar to Fig. 3d (i.e. one-step bioleaching), with small particles on the surface of the hyphae within the fungal pellet. Precipitation of particles on the surface of the hyphae both within and outside the pellet is evident. EDX analysis (Fig. 4c) shows results similar to Fig. 3e and confirmed that the particles were composed of calcium, carbon and oxygen. This is supported by the XRD spectrum (Fig. 4a) which shows evidence of calcium Anacetrapib oxalate precipitation on Day 7. SEM photomicrographs on Day 8, Day 17, and Day 27 (data not shown) in two-step bioleaching were similar to Fig. 4b. EDX results showed that the fungal pellet on Day 8, Day 17, and Day 27 contained carbon, oxygen and calcium (data not shown), similar to Fig. 3e. XRD spectra on Day 8, Day 17 and Day 27 (Fig. 4a) shows that the peak pattern in the crystal structure matched that of calcium oxalate hydrates. Although the mechanism of calcium oxalate precipitation was similar in both one-step and two-step bioleaching, the dissolution rate of fly ash was different. XRD results at Day 7 in one-step bioleaching (Fig. 3f) confirmed the presence of fly ash particles. However, XRD results at Day 7 in two-step bioleaching (Fig. 4a) shows the absence of fly ash.