A fast and simple method to estimate relative, hyphal tensile-strength of filamentous fungi used to assess the effect of autophagy

Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. Until now, there is not an efficient method which allows its characterization. Currently available methods are very time consuming; thus, compromising their applicability in strain selection and process development. To overcome this issue, a method for fast and easy, statistically-verified quantification of relative hyphal tensile strength was developed. It involves off-line fragmentation in a high shear mixer followed by quantification of fragment size using laser diffraction. Particle size distribution (PSD) is determined, with analysis time on the order of minutes. Plots of PSD 90^th percentile versus time allow estimation of the specific fragmentation rate. This novel method is demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy for Aspergillus nidulans (paternal strain) and a mutant strain (ΔAnatg8) lacking an essential autophagy gene. Both strains were grown in shake flasks, and relative hyphal tensile strength was compared. The mutant strain grown in control conditions appears to be weaker than the paternal strain, suggesting that Anatg8 may play a role in other processes involving cell wall biosynthesis. Furthermore, rapamycin-induced autophagy resulted in apparently weaker cells even for the mutant strain. These findings confirm the utility of the developed method in strain selection and process development.