Heterogeneity of filamentous growth under varying shear-force regimes

Aspergillus niger and other filamentous organisms are important producers of numerous bioactive substances, organic acids and proteins. The formation of different fungal macromorphologies, such as dispersed mycelium or pellet structures, is a multifactorial process that is difficult to control, depends largely on the cultivation conditions and influences product formation. Although heterogeneities within populations have been known for a long time, the genetic basis and ways of rationally intervening in the process have not yet been systematically investigated. Also, cell-cell and cell-bioreactor interactions for filamentous fungi are poorly understood, and the evolution of mono-species and multi-species macromorphologies, as well as their influence on the product portfolio of A. niger (citric acid, glucoamylase, enniatin B) and the formation of new natural products in co-cultivation with Streptomyces coelicolor, new experimental platforms were recently applied (2-dimensional rocking-motion single-use bioreactor) and different macromorphologies were deliberately induced by the addition of particles.

The influence of the different cultivation conditions on growth, physiology, morphology and product formation of A. niger was investigated using various online and offline microscopy techniques, viability analyses and metabolome analyses as well as X-ray microcomputed tomography. For a more intensive analysis, A. niger strains were developed that secrete the secretory protein glucoamylase (GlaA) in addition to the secondary metabolite enniatin B under control of the Tet-on system. The strains expressing eGFP and dTomato (green/red) were used to study the effects of initial spore number on macromorphological formation in shaking cultures. A µCT-based method was adapted for the detailed investigation of structures and a 3D image analysis was further developed. It was demonstrated that morphologic control (dispersed growth, fluffy or dense pellet formation) under a low-shear force enviroment becomes possible in a rocking-motion bioreactor. It was further demonstrated that the initial macromorphology had a huge impact on the subsequent cultivation performance also under typical stirred tank reactor conditions (similar toindustrial scale). Now, the knowledge is used to identify an optimal workflow for the ssed train cultivation and a better understanding of the underlying principles for controling the macromorphology.