Spore inoculum quality in filamentous bioprocesses is a critical parameter associated with viable spore concentration (1) and spore germination (2). It influences pellet morphology and, consequently, process performance. The state-of-the-art method to measure viable spore concentration is tedious, associated with significant inherent bias, and not applicable in real-time. Therefore, it is not usable as process analytical technology (PAT). Spore germination has so far been monitored using image analysis, which is hampered by complex medium background often observed in filamentous bioprocesses. The method presented here is based on the combination of viability staining and large-particle flow cytometry which enables measurements in real-time and hence aims to be applicable as a PAT tool. It is compatible with the complex media background and allows the quantification of metabolically active spores and the monitoring of spore germination. A distinction of germinated spores and not germinated spores was based on logistic regression, using multiparameteric data from flow cytometry. In a first step, a significant correlation between colony-forming unit (CFU) counts and viable spore concentration (1) in an industrially relevant model bioprocess was found. Spore germination (2) was followed over the initial process phase with close temporal resolution. The validation of the method showed an error below 5 %. Differences in spore germination for various spore inocula ages and spore inoculum concentrations were monitored. The real-time applicability of the method suggests the implementation as a PAT tool in filamentous bioprocesses.
Keywords: Filamentous fungi; Flow cytometry; Process analytical technology; Spore germination; Spore viability.