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This paper describes the role of nutrients in the production of rifamycin B by Amycolatopsis mediterranei S699 in optimal medium. The central composite design experiments showed that optimal concentrations of glucose and ammonium sulfate were 63 g/L and 7.9 g/L at 10 g/l of corn steep liquor (CSL). A bioreactor culture was performed in optimal medium. The total amino acids derived from CSL were completely depleted and 50% of the ammonium ion was used in lag phase. Cell growth was concomitant with uptake glucose and inorganic phosphate and ceased due to depletion of inorganic phosphate. Rifamycin B production began when the cell growth started, however, its production rate increased after the cell growth stopped. During this period, protein derived from CSL hydrolyzed to free amino acids which were then utilized and
simultaneous utilization of ammonium ions was observed. Production stopped at 172 h when the glucose was depleted. The maximum cell concentration and rifamycin B titer were 8.6 g/L and 1260 mg/L, respectively. This result implied that glucose and ammonium sulfate contributed to cell growth and product formation whereas CSL was found to enhance the production rate and increasing rifamycin B titer.