AbstractMost industrial production processes are performed in fed‐batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed‐batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild‐type strain (DSM 70277) and a recombinant strain pC10‐FMD (PFMD‐GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH‐drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow‐release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38–0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6‐MES mineral medium. In fed‐batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420‐fold improvement in fed‐batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose. © 2006 Wiley Periodicals, Inc.