Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane‐based fed‐batch shake flasks

Abstract

To overcome catabolite repression, industrial fermentation processes are usually operated in substrate-limited fed-batch mode. Therefore, the implementation of such an operating mode at small scale is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well-known producer of proteases, was cultivated with carbon (glucose)-and nitrogen (ammonium)-limited fed-batch conditions using the previously introduced membrane-based fed-batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5-and 2.1-fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR)signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed-batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (Y P/Glu ). In addition to Y P/Glu , protease yields were determined based on the metabolized amount of oxygenThe results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membranebased fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions. K E Y W O R D SBacillus licheniformis, catabolite repression, fed-batch, membrane-based fed-batch shake flask, proteases

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

“…Precultures were divided in two steps, whereby the first preculture was carried out in complex TB medium and the second in V3 mineral medium. A detailed description of the RAMOS‐based two‐step preculture procedure can be found in Habicher et al …”

“…For fed‐batch main cultivations, no initial glucose was supplemented to the V3 mineral medium. A detailed description of the preparation of the membrane‐based fed‐batch shake flasks can be found in literature …”

“…Offline samples were analyzed regarding the optical density at 600 nm (OD 600 ), the pH‐value, and the protease activity. A detailed description of the applied procedures can be found in Habicher et al . The protease activity measurement is based on the method developed by DelMar et al.…”

“…This process mode is predominantly used for large‐scale protease production . The positive influence of substrate‐limited fed‐batch conditions compared to batch conditions in terms of protease yields was recently affirmed with a protease producing Bacillus licheniformis strain …”

“…Only in recent years, techniques were developed to realize continuous feeding in small‐scale shaken bioreactors using microfluidic systems, diffusion‐based systems, and enzymatic release systems . The applicability of such systems, however, also depends on low investment and operating costs, compatibility with existing laboratory equipment, versatility, and reproducibility .…”