To explore the saline-alkali resistance, growth of A. niger and P. oxalicum was evaluated at NaCl concentrations of 5%, 10%, 15%, and 20% (w/v) and at pH values of 5.7 (control), 7, 8, 9, 10, 11, and 12. Modified Vogel’s minimal medium supplemented with 1.5% sucrose and 1.5% agar was used for pH and NaCl tolerance assays (Metzenberg, 2003). A sterilized dialysis membrane (Sigma-Aldrich) was placed over the agar to facilitate fungal removal and determine pH (Vergara-Fernández, Hernández, Martín-Davison, & Revah, 2011). 2.4 Bauxite residue tolerance tests BR samples were dried at 60 °C to constant weight. The fraction <212 µm, obtained using a #70 test sieve (ASTM E11), was stored at 4 °C. BR-Vogel’s medium plates for determining A. niger and P. oxalicum tolerance were prepared with increasing residue concentrations: BR-A and BR-C ranged from 5% to 40% (w/v) in 5% increments, BR-B ranged from 2% to 10% (w/v) in 2% increments (Table 8). Table 8- Initial pH of BR-Vogel’s medium plates, and estimated sodium chloride content (as halite). Sample Parameter Bauxite residue concentration 0 2 4 5 6 8 10 15 20 25 30 35 40 Control pH 5.7 NaCl % - BR-A pH 7.4 7.6 7.7 7.8 8.3 8.4 8.5 8.6 NaCl % 0.09 0.18 0.27 0.36 0.45 0.54 0.63 0.72 BR-B pH 7.7 8.0 8.4 8.6 8.8 NaCl % - - - - - BR-C pH 7.7 7.9 8.2 8.4 8.6 8.7 8.7 8.9 NaCl % 0.59 1.18 1.78 2.37 2.96 3.55 4.14 4.74 2.5 Inoculation and growth measurement Solid cultures were inoculated with 1 × 10⁵ spores from spore suspensions of A. niger and P. oxalicum by placing them on a sterile inoculation disk positioned at the center of the plate. Plates were incubated in the dark at 30 °C. Linear growth was evaluated by measuring colony diameter daily. A. niger cultures were monitored for 11 days to assess pH, salinity, and BR tolerance, whereas P. oxalicum cultures were evaluated for 18 days for BR tolerance and 11 days for pH and salinity tests. The tolerance index (TI) was calculated by dividing the diameter of fungal growth under the tested condition by the diameter of growth on the control plate, following previously described methodology (Table 9) (Rose & Devi, 2018). Mean growth diameters across conditions within each Vogel’s minimal agar treatment were compared using one-way ANOVA, with post-hoc significance assessed by Tukey’s HSD test ( = 0.05).
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