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

Abstract

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.

Catalytically Active Inclusion Bodies─Benchmarking and Application in Flow Chemistry

“…For rapid screening purposes, we employed automated microscopy, on live cells overproducing the different fusion constructs. All TdoT reference strains showed sustained IB/CatIB formation (Figure A–C, rightmost panels), in agreement with the literature. ,, This clearly proves the applicability of the automated microscopy method for rapid detection of IB/CatIB formation.…”

Understanding and controlling filamentous growth of fungal cell factories: novel tools and opportunities for targeted morphology engineering

“…For the description of the morphological development of small mycelia, powerful lattice-free [ 26 ] or lattice-based methods [ 27 ] are available today, both of which most often do make direct use of biological details but focus only on more fundamental (transport) processes and tip extension and branching. For obtaining calibration data, methods of (time lapse) image analysis are routinely applied [ 22 ], and are even coupled to high-throughput cultivation systems [ 28 ]. On the level of larger mycelia and pellets, growth and branching rates, and thus, structural properties, are additionally affected by process conditions such as substrate availability and broth rheology.…”

Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi

“…Automatized engineering of protoplasted filamentous fungi using microtiter plates and robotic liquid handling robots have been successfully established (Kuivanen et al, 2019), as well as fully-automated microscale bioreactor cultivations (Jansen et al, 2021), but working with BGC-coding DNA is demanding. The large size, the numerous genes, and the costs for total cluster DNA synthesis are limiting factors for rapid assembly and screening of numerous BGCs.…”