Abstract
<title>Abstract</title> <p> Azomycin (2-nitroimidazole), a compound synthesized by <italic>Streptomyces eurocidicus</italic> , is medically significant as a build block for the drug applied in treatment for drug-resistant tuberculosis and Chagas disease, as well as tumor hypoxia imaging. Despite its clinical value, the optimized bioprocess parameters for its production remain largely unexplored. This study evaluated the influence of various carbon and nitrogen sources (specifically glucose, starch, soybean oil, and casamino acids) on azomycin yields during submerged cultivation. Batch fermentations revealed that azomycin biosynthesis is phase-shifted, initiating only after the primary carbon source is near depletion. Notably, although soybean oil served as an excellent carbon source for biomass accumulation, it yielded no detectable azomycin production. By contrast, supplementing the media with free amino acids (casamino acids) substantially enhanced product synthesis. Using an optimized semi-defined medium of glucose and casamino acids, a titer of 46.34 mg/L was achieved in mini-bioreactors. Scaling this configuration to an instrumented 3L bioreactor resulted in a final concentration of 69.18 mg/L over 7 days, marking an 89% increase in the product-to-biomass yield Y <sub>p/x</sub> compared to smaller scales. Liquid chromatography-mass spectrometry (LC-MS) verified the identity of the extracted 2-nitroimidazole. These findings demonstrate that the precision balancing of glucose and organic nitrogen is essential to unlock and maximize the biosynthetic potential of wild-type <italic>S. eurocidicus</italic> . </p>