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Most 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. This may lead to wrong strain selections. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation based on diffusion in shaken bioreactors. No other device for feeding was required. This “slow-release fed-batch technique” was tested on the metabolism of H. polymorpha, E. coli and G. oxydans in shake flasks. The OTR and RQ were monitored online with a RAMOS device. Biomass formation, synthesis of proteins like GFP or eYFP-IL-6, pH drift and metabolic dynamics of glucose, ethanol, acetic acid and other organic acids were measured offline. By application of the slow-release fed-batch technique in comparison to regular batch mode, overflow-metabolism of H. polymorpha and E. coli could be reduced, which led to an increase in biomass yield of up to 85% and 59%, respectively. Up to date, 23.4 g/L cell dry weight of H. polymorpha and 13.7 g/L of E. coli was achieved. The specific biomass yields of 0.38-0.47 are in the magnitude of those in laboratory fermentors equipped with a substrate feed-pump. The GFP expression by H. polymorpha RB11 pC10-GFP could be improved in Syn6-MES and YNB mineral media up to 35-fold and 420-fold, respectively. The synthesized maximum in fed-batch mode was 421 mg/L GFP. In contrast only up to 2.5 mg/L GFP was received in batch mode. The expression of eYFP-IL-6 by E. coli BL21 pLys pRSET eYFP-IL6 could be increased 4-fold in optimized Wilms-MOPS mineral medium using the slow-release fed-batch technique. Slow-release fed-batch cultures of G. oxydans DSM 2003 revealed a 2.6-fold increase of specific biomass yield in modified Silberbach-MES complex medium. A cell dry weight of 3.3 g/L was obtained in contrast to 1.6 g/L in batch mode. Due to glucose feeding a reduction of gluconic acid as well as 2- and 5-ketogluconic acid formation was monitored. A mass screening of 265 H. polymorpha RB11 pC10-FMD clones and 267 pC10-MOX clones was performed in Syn6-MES mineral medium in deep-well plates. A batch with glucose and one with glycerol were performed simultaneously in comparison to a slow-release fed-batch with glucose. One repetition screening was done under the same conditions. These diverse operational modes revealed great differences in strain selection and quality of specific GFP yield. The best strains for a fed-batch would be unlikely found in either batch mode. A dependence on the carbon source and the operational mode was found in relevance to the regulating promoter for gene expression. A fed-batch screening points out to be the most secure way to select the right strain for a fed-batch production process.