The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering

Fungi significantly influence biogeochemical processes, particularly in aerobic terrestrial environments where they acquire nutrients through the breakdown of organic matter and the weathering of rock substrates. Rock-inhabiting black fungi, such as *Knufia petricola*, are ubiquitous colonizers of exposed mineral surfaces and form biofilms that contribute to weathering and soil formation. These fungi produce extracellular polymeric substances (EPS) and the dark pigment DHN melanin, both of which offer protection against various environmental stresses. While the protective roles of EPS and melanin are known, their contribution to mineral weathering remains ambiguous. This study focuses on the mechanistic understanding of the interaction between *K. petricola*, its EPS, and olivine dissolution.

The literature review extensively cites previous research on fungal-mineral interactions, the role of fungi in biogeochemistry and geobiology, the importance of microbial biofilms, and the characteristics of rock-inhabiting black fungi. Existing knowledge on EPS composition (proteins, lipids, polysaccharides, DNA) and its functions in biofilm stability, protection against desiccation and toxic substances, and nutrient accumulation is summarized. However, the role of EPS in weathering processes remains debated in the literature, with some studies suggesting both enhancement and inhibition depending on the specific context.

The study utilized four pigment mutants of *K. petricola* (Δpks1, Δsdh1, Δphd1, and Δpks1/Δphd1) with varying melanin and carotenoid production capabilities. Fungi were cultivated as subaerial biofilms, and EPS was extracted and characterized using various techniques, including weight quantification, general chemistry analysis (GC-MS, XPS, FTIR), and monosaccharide composition determination. Biofilm and EPS morphology were visualized using cryo-scanning electron microscopy (cryo-SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM). Batch olivine dissolution experiments were conducted to measure the dissolution rates of olivine in the presence of different *K. petricola* strains. Mg, Si, and Fe concentrations were analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Lectin screening was used to investigate the binding capacity of lectins to glycoconjugates in *K. petricola* biofilms. Statistical analysis using ANOVA with Tukey-HSD post hoc test and Pearson's correlation test was performed to evaluate differences in means and correlations among the measured variables.

Melanin-deficient mutants (Δpks1, Δpks1/Δphd1) produced significantly more EPS than the wild type (WT) and the carotenoid-deficient mutant (Δphd1). The EPS of the melanin-deficient mutants showed a different monosaccharide composition with higher mannose and galactose and lower glucose content compared to the WT. FTIR analysis revealed shifts in characteristic carbohydrate bands, consistent with changes in polysaccharide composition. The melanin-deficient mutants had significantly lower grades of attachment to olivine and lower olivine dissolution rates compared to the WT and Δphd1. The Δsdh1 mutant, which accumulates melanin precursors, exhibited intermediate biofilm thickness, EPS production, and olivine dissolution rates. The grade of attachment to olivine correlated positively with the fraction of pullulan-related linkages in the EPS. The Δsdh1 mutant, despite having an intermediate attachment, displayed the highest olivine dissolution rate. This was linked to the release of water-soluble melanin precursors, which possibly act as Fe-chelating agents, preventing Fe oxidation and thereby enhancing dissolution.

The findings demonstrate a clear link between melanin production, EPS composition, fungal attachment to olivine, and the rate of olivine dissolution. The higher EPS production in melanin-deficient mutants suggests a compensatory mechanism for the lack of melanin's protective functions. The correlation between pullulan-related linkages in EPS and olivine attachment suggests a crucial role of these polysaccharides in adhesion. The enhanced dissolution rate observed in the Δsdh1 mutant points to the potential of melanin precursors as Fe-chelating agents, contributing to olivine weathering independently of attachment. The results highlight the complex interplay between biotic and abiotic factors in mineral weathering processes.

This study shows that EPS composition and fungal attachment to the substrate are critical for bioweathering. Melanin production influences EPS quantity and composition, impacting the ability of the fungus to attach and dissolve olivine. Pullulan-related linkages in EPS appear crucial for attachment, and melanin synthesis intermediates can act as effective Fe chelators, further stimulating olivine dissolution. Future research should investigate the specific roles of other EPS components and further explore the mechanisms underlying the chelating activity of melanin precursors.

The study was conducted under controlled laboratory conditions, and the results may not fully reflect the complexity of natural environments. The batch reactor design might have introduced limitations due to nutrient depletion and metabolite accumulation over time. The relatively small sample size, especially for some of the EPS analyses, may also have affected statistical power.