An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale-up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R-o of 68 vs. 92 min(-1)) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm(2)) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40-50% decrease). High off-gas CO2 levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25-fold scale-up (600-15,000 L) was controlled to match off-gas CO2 profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L scale fermentation achieved an expected nocathiacin 1 titer of 310 mg/L after 7 days. Other on-line data (i.e., pH, oxygen uptake rate, and CO2 evolution rate) and off-line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale-up. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 176-188, 2009