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

Abstract

Filamentous fungi are exploited as cell factories in biotechnology for the production of proteins, organic acids, and natural products. Hereby, fungal macromorphologies adopted during submerged cultivations in bioreactors strongly impact the productivity. In particular, fungal pellets are known to limit the diffusivity of oxygen, substrates, and products. To investigate the spatial distribution of substances inside fungal pellets, the diffusive mass transport must be locally resolved. In this study, we present a new approach to obtain the effective diffusivity in a fungal pellet based on its three-dimensional morphology. Freeze-dried Aspergillus niger pellets were studied by X-ray microcomputed tomography, and the results were reconstructed to obtain three-dimensional images. After processing these images, representative cubes of the pellets were subjected to diffusion computations. The effective diffusion factor and the tortuosity of each cube were calculated using the software GeoDict. Afterwards, the effective diffusion factor was correlated with the amount of hyphal material inside the cubes (hyphal fraction). The obtained correlation between the effective diffusion factor and hyphal fraction shows a large deviation from the correlations reported in the literature so far, giving new and more accurate insights. This knowledge can be used for morphological optimization of filamentous pellets to increase the yield of biotechnological processes.

Growing a circular economy with fungal biotechnology: a white paper

“…Fourier-transformed infrared spectroscopy has become a next-generation phenotyping technology to identify fungal strains and their metabolic products [134], while the power of surface analysis tools, such as mass spectrometry-based imaging techniques, has been exploited to identify changes that are brought about by filamentous fungi and their enzymes when they attack and modify insoluble lignocellulose materials [135]. "Ready-touse-microfluidics" have been implemented to study the dynamics of fungal growth and fungal interaction with soil bacteria [136], X-ray microcomputed tomography has been successfully applied to study spatial distribution of hyphae within mycelia and diffuse mass transport therein [137,138], and gene and protein networks have been developed to assist our understanding of filamentous fungal biology holistically [128,139]. Hence, the technological foundation is sound and powerful and no longer represents a critical hurdle for gaining knowledge on fungal biology.…”

Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry

“…Even more comprehensive understanding of the morphology's impact on substrate transport and viability in pellets can be obtained through modeling or experimental approaches. To model the transport of substrates into pellets, their detailed micro-morphology has to be known, which was shown for filamentous fungi (Aspergillus niger) with hyphal diameters > 3 μm [17]. Experimentally, a combination of viability staining and confocal laser-scanning microscopy (CLSM) facilitates the distinction between active and inactive regions inside individual pellets off-line [10].…”

Universal law for diffusive mass transport through mycelial networks

“…Notably, the metabolic rate could be estimated if the first two prerequisites and the concentration profile are known. We, therefore, recently harnessed X‐ray microcomputed tomography (µCT) and three‐dimensional (3D) image analysis to determine the location and number of tips, branches, and hyphal material within whole fungal pellets to meet the first two requirements (Schmideder, Barthel, Friedrich, et al, 2019; Schmideder, Barthel, Müller, et al, 2019). Mathematically, the concentration profile of any molecule within the voids of a pellet could be described by a diffusion reaction equation (Celler et al, 2012; Meyerhoff et al, 1995), where the reaction term describes the consumption or the production of the molecule.…”

“…It has to be mentioned that is similar to the formation factor, which is often applied to describe the diffusivity, conductivity, or permeability through porous media (Tomadakis & Robertson, 2005). In our recent study (Schmideder, Barthel, Müller, et al, 2019), we proposed the preliminary correlation for one of the main fungal cell factories, A. niger , where is the hyphal fraction (equal to solid fraction).…”

“…In the current study, we investigated the correlation between the effective diffusivity and the morphology of more than 60 µCT measured pellets from four fungal cell factories ( A. niger, P. chrysogenum, Rhizopus oryzae , and R. stolonifer ) and more than 3000 Monte Carlo simulated pellets. We considered the diffusion through filamentous networks with different resolutions because image resolution can influence computed material properties (Schmideder, Barthel, Müller, et al, 2019; Velichko et al, 2009). Based on the measurements and simulations, we propose here a generalized law for the diffusion of nutrients, oxygen, and fungal metabolites through hyphal networks.…”