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As Streptomycetes might produce melanin to survive in stressful environmental conditions, like under metal exposure, supplementing metal ions to the growth medium could be a wise strategy for boosting the production of the pigment. The aim of this study was to test, for the first time, the possibility of boosting S. nashvillensis DSM40314 melanin biosynthesis by adding to the growth medium singularly or, at the same time, different concentrations (1.0, 1.5, and 2.0 g∙L−1) of CuSO4 or/and Fe2(SO4)3. A maximum melanin production of 4.0 ± 0.1 g·L−1 was obtained in shake flasks with a 2.0 g∙L−1 coupled addition of the two metals, while the extracellular tyrosinase activities ranged values between 5.4 and 11.6 ± 0.1 U·L−1. The pigments produced in different conditions were precipitated from the broth supernatants under acidic conditions, purified, and characterized by UV-VIS, FT-IR, and NMR analyses that determined structures like eumelanin pigments. Fermentation experiments in stirred tank reactors allowed to scale up the process in more controlled conditions, further boosting the pigment production up to 4.9 ± 0.1 g·L−1, with an increase of about 22.0% compared to the results obtained in shake flasks. [ABSTRACT FROM AUTHOR]