730 2013; Toli et al., 2023; Ujaczki et al., 2017). In recent years, the valorization of secondary sources through bioleaching has gained attention for its potential to recover critical metals through their solubilization, while also providing an economical and environmentally friendly strategy for the remediation of solid industrial waste (Gu, Rastegar, Mousavi, Li, & Zhou, 2018). The bioleaching of secondary sources using versatile and resilient fungal strains has been studied for their efficiency in metal recovery and neutralization through organic acid production. They have shown significant potential, as they can thrive in harsh environments with elevated salinity and alkalinity, which are main characteristics of BR, neutralize its alkalinity, and enhance metal recovery and pollutant chelation. Moreover, these strains can utilize agricultural solid wastes as a carbon source, with their metabolism-associated organic acid production contributing to metal solubilization and improving the economic feasibility of the process (Egbe, Ihediwa, Abdulsalami, & Adebayo, 2022; Zhang et al., 2020). This research aims to assess the tolerance of Aspergillus niger and Penicillium oxalicum, to BR, alkalinity and high‑salinity conditions to determine their potential for neutralizing and remediating this waste. 2. MATERIALS AND METHODS 2.1 Bauxite Residue Samples Four BR samples were collected from two alumina refineries in Australia after the Bayer process and were previously chemically and mineralogically characterized (Soto-Montandon et al., In preparation). BR-A and BR-C were seawater-neutralized, whereas BR-B remained untreated (Table 11). Table 11- BR samples, refinery of origin, the year of collection, post-treatment method and pH. Sample Refinery Year of collection Treatment pH BR-A A 2024 Seawater 9.6 BR-B A 2023 None 11.3 BR-C B 2010 Seawater 9.7 2.2 Fungal Strains And Spore Preparation A. niger (DSM 2466) and P. oxalicum (DSM 898) were obtained from DSMZ and reactivated following DSMZ protocols (The Leibniz Institute). Spore suspensions were prepared by incubating cultures on Potato Dextrose Agar at 30 °C for 7 days. Spores were harvested with 1% Triton X-100, filtered to remove mycelium, and adjusted to ~2 × 106 spores/mL using a hemocytometer. Suspensions were stored at -80 °C. 2.3 Ph And Salinity Tolerance Tests 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 12).
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