Figure 25- pH variation of the culture medium after 11 days of incubation with (A) A. niger and (B) P. oxalicum in different alkalinity and salinity conditions. Values are expressed as ΔpH ( - ). Negative ΔpH values represent a reduction in pH (acidification) relative to the initial medium. Means and confidence intervals (95%) are presented. 3.2 Effect of BR on the growth of the selected fungal strains The tolerance of filamentous fungi to BR is a critical determinant of their suitability for both bioleaching and bioremediation applications, and our results demonstrate strong residue-specific and strain-specific responses. A. niger tolerated BR concentrations up to 20% (w/v) in BR-A, 2% (w/v) in BR-B, and 25% (w/v) in BR-C, with growth inhibition increasing with the concentration (Figure 26). These tolerance limits align with previous reports indicating a maximum BR tolerance of approximately 10% (w/v) for this strain (Kiskira et al., 2023; Qu et al., 2015; Reddy, 2025). Although the initial pH values at limiting concentrations for BR-A, BR-B, and BR-C were below previously established pH tolerance thresholds (Figure 23.A), inhibition persisted, demonstrating that pH alone does not govern fungal survival. Moreover, despite BR A and BR C showing similar pH after seawater neutralisation (Table 8), their differing inhibition patterns highlight the influence of other physicochemical factors, including ionic strength, exchangeable sodium percentage, and multi-metal interactions, on fungal tolerance. While most metals were present at levels well below the reported minimal inhibitory concentrations, iron concentrations exceeded these values, ranging from 3,949 to 10,734 ppm, compared with inhibitory concentrations of 2,000 ppm and 1,117 ppm for A. niger and P. oxalicum, respectively (Anahid, Yaghmaei, & Ghobadinejad, 2011; Levinskaitė et al., 2009). The known synergistic inhibitory effects of multi metal exposure (Le, Tang, Ryan, & Valix, 2006; Tzeferis, 1994; Valix & Loon, 2003) likely contributed to the reduced tolerance observed at higher BR concentrations. Despite the BR stress, A. niger showed substantial neutralisation capability, decreasing BR surface pH by 0.6-2.2 units, values consistent with earlier reports for submerged fermentations, where pH reductions of 1.5-2.5 units were observed at pulp densities of 1-10% (w/v) (Kiskira et al., 2023), and demonstrating the capability to decrease pH from 10 to 3.6. Such strong organic-acid production is essential for both proton-driven mineral dissolution in bioleaching and the neutralisation of alkaline residues during bioremediation. P. oxalicum exhibited lower tolerance overall, with maximum tolerated concentrations of 15% (BR-A), 2% (BR-B), and 20% (BR-C), with increased lag phases and growth inhibition. A) B)
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