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

Abstract

Introducing fed-batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed-batch fermentation processes. Therefore, cost efficient and easy to use small-scale fed-batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer-based controlled-release fed-batch microtiter plate is described. In this work, the polymer-based controlled-release fed-batch microtiter plate is used to investigate fed-batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online-monitored within each well of the polymer-based controlled-release fed-batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer-based controlled-release fed-batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed-batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed-batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale-up criterion, microtiter plate-and shake flask-based fed-batch cultivations are highly comparable. On basis of the small-scale fed-batch cultivations, a mechanistic model is established and validated. Model-based simulations coincide well with the experimentally acquired data.

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

“…Due to the release principle of this microtiter plate (Fig. 1 ), glucose is released from the beginning of the cultivation (Additional file 3 ) [ 26 , 27 ]. At this point, the biomass concentration is too low to consume all of the released glucose.…”

“…This results in an initial batch phase. Its length is dependent on the initial biomass concentration [ 27 ]. Low initial biomass concentrations might cause an extended batch phase with oxygen limitation and overflow metabolism.…”

“…Cultivations were performed with a shaking frequency n = 1000 rpm, a shaking diameter d 0 = 3 mm, at 30 °C and a relative humidity φ > 75%. The cultivation conditions in microtiter plate-based batch and fed-batch main cultures were adjusted so that an oxygen limitation was prevented (Additional file 2 ) [ 27 ]. Culture acidification was prevented by using MOPS buffer, thereby keeping the pH in the neutral range between 7 and 7.5 (Additional file 2 ) [ 27 ].…”

“…The cultivation conditions in microtiter plate-based batch and fed-batch main cultures were adjusted so that an oxygen limitation was prevented (Additional file 2 ) [ 27 ]. Culture acidification was prevented by using MOPS buffer, thereby keeping the pH in the neutral range between 7 and 7.5 (Additional file 2 ) [ 27 ]. Batch and fed-batch cultivations were terminated after 65 and 72 h, respectively.…”

“…In this study, the previously established fed-batch process for protease expression with B. licheniformis in fed-batch MTP’s [ 27 ] was applied to perform a high-throughput and glucose-limited fed-batch screening campaign. The screening campaign was performed with a full set of B. licheniformis clones from two strain lineages without pre-selection (13 in total).…”