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

Biotechnol J. 2020 Feb;15(2):e1900088. doi: 10.1002/biot.201900088. Epub 2019 Sep 19.


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.

Keywords: Bacillus licheniformis; fed-batch; microtiter plate; protease; shaken bioreactors.

MeSH terms

  • Bacillus licheniformis / enzymology*
  • Bacillus licheniformis / genetics
  • Bacillus licheniformis / growth & development
  • Batch Cell Culture Techniques / methods*
  • Biomass
  • Bioreactors
  • Culture Media
  • Fermentation
  • Glucose / metabolism*
  • Industrial Microbiology / methods*
  • Osmotic Pressure
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / metabolism*
  • Polymers
  • Reproducibility of Results


  • Culture Media
  • Polymers
  • Peptide Hydrolases
  • Glucose