Abstract
BackgroundMorphology, 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.ResultsIn 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.ConclusionsA 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.
Highlights
Morphology, being one of the key factors influencing productivity of filamentous fungi, is of great interest during bioprocess development
As a starting point for A. carbonarius BioLector-assissted microtiter plate cultivation, similar conditions as recently introduced in Jansen et al [22] for A. giganteus were tested, i.e. 600 rpm in a Round Well Plate cultivated in Sinha medium with 2 % (w v−1) CaCl2
A medium previously described for A. carbonarius MTP cultivation by Linde et al [23], with addition of 2 % (w v−1) CaCl2, was tested (Fig. 1a)
Summary
Morphology, being one of the key factors influencing productivity of filamentous fungi, is of great interest during bioprocess development. The process development and cultivation of filamentous fungi remains challenging due to their complex morphological changes during submerged cultivation ranging from dispersed mycelia to millimetersized pellet morphology [3, 4]. Larger aggregates and pellets are beneficial for mass transfer and simplify downstream processing due to unproblematic biomass removal via filtration [11]. This state of morphology might be disadvantageous because of diffusion limitation into the dense core region of large pellets, which as a result might lead to oxygen-limited cells and autolysis, reducing productivity [5]. For each production process the desired morphology remains highly specific as a key process parameter impacting productivity [4, 12]
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