Abstract
Filamentous fungi serve as production host for a number of highly relevant biotechnological products, like penicillin. In submerged culture, morphology can be exceptionally diverse and is influenced by several process parameters, like aeration, agitation, medium composition or growth rate. Fungal growth leads to several morphological classes encompassing homogeneously dispersed hyphae and various forms of hyphal agglomerates and/or clump structures. Eventually, the so-called pellet structure can be formed, which represents a hyphal agglomerate with a dense core. Pellet structures can hinder oxygen and substrate transport, resulting in different states of viability, which in turn affects productivity and process control. Over the years, several publications have dealt with methods to either gain morphological insight into pellet structure or determine biomass viability. Within this contribution, we present a way to combine both in a flow cytometry–based method employing fluorescent staining. Thereby, we can assess filamentous biomass in a statistically sound way according to (i) morphology and (ii) viability of each detected morphological form. We are confident that this method can shed light on the complex relationship between fungal morphology, viability and productivity—in both process development and routine manufacturing processes.
Highlights
Successful cultivation strategies involving filamentous fungi need to consider the organism’s morphology
We present a novel combination of morphological analysis and viability assessment based on flow cytometry
Enhanced insight into viability is generated simultaneously through fluorescent staining: Overall viability, viability of morphological classes and the viable layer of large elements can be determined. This viability data is enhanced by morphological parameters like pellet and large element compactness
Summary
Successful cultivation strategies involving filamentous fungi need to consider the organism’s morphology. Fluorescent staining is regularly used in flow cytometry coupled with fluorescence detectors (Langemann et al 2016; Attfield et al 2000; Pekarsky et al 2018) For filamentous fungi such studies are scarce, mainly due to the large particle sizes of fungal biomass (Dubelaar et al 1999). 2 μl of freshly prepared 50 mg/l FDA reagent (Sigma-Aldrich, St. Louis, Missouri, USA; prepared with PBS buffer from a stock solution of 5 g/l dissolved in acetone) was added and a cover slide was placed on the sample. Pictures were taken for at least 10 pellets per sample
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