Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry—the fast alternative to microscopic image analysis

Abstract

An important parameter in filamentous bioreactor cultivations is the morphology of the fungi, due to its interlink to productivity and its dependency on process conditions. Filamentous fungi show a large variety of morphological forms in submerged cultures. These range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the objective function of the bioprocess, different characteristics of the morphology are favorable and need to be quantified accurately. The most common method to quantitatively characterize morphology is image analysis based on microscopy. This method is work intensive and time consuming. Therefore, we developed a faster, at-line applicable, alternative method based on flow cytometry. Within this contribution, this novel method is compared to microscopy for a penicillin production process. Both methods yielded in comparable distinction of morphological sub-populations and described their morphology in more detail. In addition to the appropriate quantification of size parameters and the description of the hyphal region around pellets, the flow cytometry method even revealed a novel compactness parameter for fungal pellets which is not accessible via light microscopy. Hence, the here presented flow cytometry method for morphological analysis is a fast and reliable alternative to common tools with some new insights in the pellet morphology, enabling at-line use in production environments.