A novel tool to determine spore inoculum quality in filamentous bioprocesses using multiparametric flow cytometry

Abstract

In this master thesis a method to determine the concentration of living spores for Penicillium chrysogenum was developed. The method is a combination of flow cytometry and fluorescence staining. The concentration of living spores can differ between spore inocula, depending on age and storage conditions. The variation of spore inoculum quality of filamentous fungi has an effect on batch-to-batch productivity. Dependent on the quality of the spore inoculum a different volume of spores has to be inoculated to achieve the optimum concentration of viable spores in the bioreactor. The common way to determine the amount of living spores is using colony formation unit determination. This method has the drawback to be time-consuming and work intensive. In this master thesis a flow cytometric method was developed and optimized to differ between living spores and medium, and further to quantify viable spores. The newly developed method was tested with series of validation experiments. Therefore, spore inocula with different concentrations of living spores were created and the concentration of each spore inoculum was determined with this method and the colony forming unit determination. Furthermore, two series of experiments were performed, where the living spore concentration of Penicillium chrysogenum in batch fermentations was monitored within the first 30 hours. In a first experiment four cultivations in 500 ml shake flasks and in a second experiment four fermentations in a 10 l bioreactor were conducted. The results of the experiments indicates a good correlation between the concentration of living spores determined with this newly developed method and the concentration obtained with the established method of colony forming unit determination. The advantage of this new method is that the results are obtained much faster and with less workload. It is also shown that the results contain much more information about the living spores than the results obtained with the colony forming unit. For example additional information about the morphology of the Penicillium chrysogenum spores can be extracted. The performance of the developed method could be proven with validation experiments and the successful application of the method was demonstrated in detail.