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Background  Morphology, being one of the key factors influencing productivity of filamentous fungi, is of great interest during bioprocess development. With increasing demand of high-throughput phenotyping technologies for fungi due to the emergence of novel time-efficient genetic engineering technologies, workflows for automated liquid handling combined with high-throughput morphology analysis have to be developed.    Results  In this study, a protocol allowing for 48 parallel microbioreactor cultivations of  Aspergillus carbonarius  with non-invasive online signals of backscatter and dissolved oxygen was established. To handle the increased cultivation throughput, the utilized microbioreactor is integrated into a liquid handling platform. During cultivation of filamentous fungi, cell suspensions result in either viscous broths or form pellets with varying size throughout the process. Therefore, tailor-made liquid handling parameters such as aspiration/dispense height, velocity and mixing steps were optimized and validated. Development and utilization of a novel injection station enabled a workflow, where biomass samples are automatically transferred into a flow through chamber fixed under a light microscope. In combination with an automated image analysis concept, this enabled an automated morphology analysis pipeline. The workflow was tested in a first application study, where the projected biomass area was determined at two different cultivation temperatures and compared to the microbioreactor online signals.    Conclusions  A novel and robust workflow starting from microbioreactor cultivation, automated sample harvest and processing via liquid handling robots up to automated morphology analysis was developed. This protocol enables the determination of projected biomass areas for filamentous fungi in an automated and high-throughput manner. This measurement of morphology can be applied to describe overall pellet size distribution and heterogeneity.

A fully automated pipeline for the dynamic at‐line morphology analysis of microscale Aspergillus cultivation