Understanding pellet population heterogeneity of Aspergillus niger in stirred tank and rocking motion bioreactors.

The filamentous fungus Aspergillus niger is a well-established cell factory in biotechnology. Its productivity depends on macromorphological development which remains difficult to control, partly because the relationship between seed culture and reactor-specific shear force conditions has not been systematically investigated. This study examined how high or low shear forces affect pellet development at both micro- and macromorphological levels in stirred-tank reactors (STR, high shear regime) and rocking-motion bioreactors (RMB, low shear regime). A. niger seed cultures with initially either large or small pellets were used to inoculate batch STR or RMB. Comparable cultivation conditions were applied so that fermentations differed mainly in shear force regime. Growth characteristics and pellet macromorphologies were analysed using 2D and 3D image analyses, enabling us to classify pellets according to three different classes based on their inner pellet architecture. The distribution of these classes depended on both the macromorphologies of the seed culture and the reactor type. Under high shear forces in the STR, pellets underwent breakage shortly after stirrer activation, were limited in their size to an average diameter of 500-600 µm, and formed a homogeneous population. In addition, broken pellets occurred predominantly under STR conditions. In contrast, cultivations in RMB preserved the initial pellet architecture, allowed the formation of larger pellets (median diameter ~ 800 µm) and supported pellet fusion, thus resulting in a more heterogeneous macromorphological population. Notably, glucose uptake rate correlated with the surface-to-volume ratio of the pellet populations, i.e., glucose became faster consumed under STR conditions accompanied with lower biomass yields and higher protein secretion. Citric acid production, however, was detectable in both STR and RMB only when reactors were inoculated with seed cultures characterised by a loose pellet morphology. Overall, our study demonstrates how shear regime and seed culture morphology jointly shape pellet architecture, population heterogeneity and productivity in scale-up processes. Such a comprehensive understanding of morphological developments is instrumental for optimising bioprocesses and future predictive modelling approaches. KEY POINTS: • 2D/3D analysis of defined seed cultures in different shear-induced environments • High shear restricts and homogenises pellets, while low shear maintains heterogeneity • Highest citric acid and total protein levels were found in smaller, compact pellets.