Secretory Vesicle and Glucoamylase Distribution in and Macromorphology in Regions of Varying Shear Stress.

In technical fermentations, filamentous microorganisms are exposed to different forms of mechanical stress, among which shear stress is prevalent in turbulent broths. Whereas small-scale bioreactors allow for realistic turbulent flow field conditions, they are not well-suited to investigate the fungal response to shear stress in more detail, as they only reveal the integral effect of a highly dynamic stress stimulus. Therefore, the widely used model system for producing constant, but rather low shear forces, the parallel plate flow chamber, is extended in this work by adding a backward-facing step (BFS). The BFS induces vortex shedding in the wake of the step and brings out distinct areas of different shear stress levels at the bottom of the chamber where mycelia grow. This allows for a stress-dependent analysis of growing cells using a confocal laser-scanning microscope. As the real stress cannot be measured in the experiment, the wall shear stress is estimated numerically using computational fluid dynamics (CFD). As a first application of the experimental setup, the relative biomass concentration, the relative amount of secretory vesicles and the relative amount of the chosen product glucoamylase produced by the filamentous fungus were measured. The obtained area scans show homogeneous mycelia growth in areas of low stress and cloud-like patterns downstream of the predicted flow reattachment length where high shear stress dominates. Quantitative analysis of the time course suggests that the amount of available secretory vesicles inside of decreases when the shear stress is increased, despite that no significant differences in biomass production could be found. In contrast, the highest level of glucoamylase was reached for intermediate volumetric flow rates, i.e., levels of shear stress.