Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis...

Area of Science:

• Biotechnology
• Microbial Physiology
• Industrial Microbiology

Background:

• Industrial fermentation often uses substrate-limited fed-batch mode to avoid catabolite repression.
• Scaling down fermentation processes is essential for maintaining consistent conditions.
• Bacillus licheniformis is a key organism for protease production.

Purpose of the Study:

• To implement and evaluate substrate-limited fed-batch conditions at a small scale.
• To investigate the impact of glucose and ammonium limitation on protease production.
• To assess the utility of membrane-based fed-batch shake flasks and Respiration Activity MOnitoring System (RAMOS).

Main Methods:

• Cultivation of Bacillus licheniformis under carbon (glucose) and nitrogen (ammonium)-limited fed-batch conditions.
• Utilized membrane-based fed-batch shake flasks for controlled nutrient feeding.
• Monitored oxygen transfer rate (OTR) using RAMOS to track nutrient status and fed-batch initiation.

Main Results:

• Protease yields increased 1.5-fold with glucose limitation and 2.1-fold with ammonium limitation compared to batch conditions.
• Elevated feeding rates increased protease activity without altering protease yield (Y_P/Glu).
• Protease production correlated with glucose consumption and oxygen metabolism.

Conclusions:

• Membrane-based fed-batch shake flasks coupled with RAMOS enable effective investigation of substrate-limited fed-batch conditions at small scale.
• This approach successfully avoids catabolite repression and enhances protease production.
• The system provides real-time feedback on nutrient status and feeding kinetics.