Influence of Diet and Growth Conditions on the Carbon and Nitrogen Stable Isotopic Composition of Aspergillus niger Mycelium: Insights for Fungal Chitosan Characterization

This study investigates, for the first time, the relationship between carbon (<i>δ</i>¹³C) and nitrogen stable isotopic composition of <i>Aspergillus niger</i> mycelium, used as chitin and chitosan sources, and the fungus diet under controlled cultivation conditions. Four diets were tested, combining different carbon (C3- and C4-glucose) and nitrogen sources (KNO₃ and NH₄Cl). Results showed that carbon sources significantly influenced <i>δ</i>¹³C values of the mycelium: C4-glucose diets led to more negative <i>Δ</i>¹³C values (<i>δ</i>¹³C_MYCELIUM-<i>δ</i>¹³C_DIET) compared to C3-glucose diets. Nitrogen sources also affected isotopic fractionation, with KNO₃ leading to negative <i>Δ</i>¹⁵N (<i>δ</i>¹⁵N_MYCELIUM-<i>δ</i>¹⁵N_DIET) and NH₄Cl yielding positive <i>Δ</i>¹⁵N. Conversely, pH and temperature showed negligible effects on <i>δ</i>¹⁵N, while continuous aeration during growth significantly decreased <i>δ</i>¹⁵N, possibly due to partial assimilation of atmospheric nitrogen. These findings demonstrate that both nutrient and cultivation parameters can modulate the isotopic fractionation in <i>A. niger</i>, particularly for nitrogen. Although a direct correlation between diet composition and <i>δ</i>¹⁵N could not be established, this work provides the first experimental link between fungal metabolism and its isotopic fingerprint. The results offer a scientific foundation for applying stable isotope ratio analysis to authenticate and trace fungal-derived chitin and chitosan, with potential applications in food and winemaking industries.