An X‐ray microtomography‐based method for detailed analysis of the three‐dimensional morphology of fungal pellets

Abstract

Filamentous fungi are widely used in the production of biotechnological compounds.Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Threedimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied-one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the threedimensional structure of pellet-forming filamentous microorganisms in utmost detail.The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications. K E Y W O R D Sfilamentous fungi, image analysis, pellets, three-dimensional morphological quantification, X-ray microtomography

From three‐dimensional morphology to effective diffusivity in filamentous fungal pellets

“…An instant adhesive (UHU, Bühl, Germany) was used to fix the freeze‐dried fungal pellets on top of a sample holder. In contrast to the previous study (Schmideder et al, ), in this case, the instant adhesive dried 5 min before placing the pellets on top of the holder. This procedure resulted in a smooth surface of the instant adhesive, while it remained sticky enough to fix the pellets.…”

“…The A. niger hyperbranching strain MF22.4, which has been shown to be a better protein‐secretion strain than the wild‐type strain (due to deletion of the racA gene; Fiedler, Barthel, Kubisch, Nai, & Meyer, ), was used in this study. Pellets were obtained by submerged cultivation of MF22.4 for 48 hr and freeze‐drying following the previously described protocol (Schmideder et al, ).…”

“…To obtain three‐dimensional images of the freeze‐dried filamentous pellets, μCT measurements were performed based on the method reported by Schmideder et al (). Two‐dimensional projections from different angles were reconstructed to generate three‐dimensional images with a custom‐designed software (Matrix Technologies, Feldkirchen, Germany) that uses CERA (Siemens, Munich, Germany).…”

“…The background included the instant adhesive used for sample fixation, the air between the hyphae, and small impurities. In general, image processing is leaned to the one reported in the section “Preprocessing” by Schmideder et al ()). The image processing result for one pellet is exemplarily illustrated in Figure .…”

“…Therefore, the appropriate correlation between pellet morphology and diffusion was not possible in this experimental approach. We could recently overcome the limitations of two‐dimensional image generation by performing X‐ray microcomputed tomography (μCT) measurements on whole fungal pellets (Schmideder et al, ). The investigated Aspergillus niger pellet showed a very complex three‐dimensional structure.…”