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Background  Expanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development.    Results  Parallelized microplate cultivation of the eukaryotic protein production host  Pichia pastoris  was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of  P. pastoris  constitutively expressing AppA phytase from  Escherichia coli . The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode.    Conclusion  The comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.    Electronic supplementary material  The online version of this article (10.1186/s40694-018-0053-6) contains supplementary material, which is available to authorized users.

Improved microscale cultivation of Pichia pastoris for clonal screening