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

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.DFG, 198187031, Mikro-Computertomograph mit integrierter Materialprüfmaschine und KühleinheitDFG, 315384307, Verallgemeinerte morphologische Modellierung aggregierender, filamentöser MikroorganismenDFG, 315305620, Untersuchung des Einflusses von Scherkräften auf das morphogenetische Gennetzwerk, die Zellintegrität, mikroskopische und makroskopische Morphologie von Aspergillus niger sowie Bildungsraten intra- und extrazellulärer Produkt

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

Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea aerocolonigenes were investigated by microscopy and flow cytometry. Both methods were compared regarding their applicability. To achieve different morphologies, a cultivation with glass bead addition (Ø = 969 μm, 100 g L-1) was compared to an unsupplemented cultivation. This led to two different macro-morphologies. Furthermore, glass bead addition increased rebeccamycin titers after 10 days of cultivation (95 mg L-1 with glass beads, 38 mg L-1 without glass beads). Macro-morphology and viability were investigated through microscopy and flow cytometry. For viability assessment fluorescent staining was used additionally. Smaller, more regular pellets were found for glass bead addition. Pellet diameters resulting from microscopy followed by image analysis were 172 μm without and 106 μm with glass beads, diameters from flow cytometry were 170 and 100 μm, respectively. These results show excellent agreement of both methods, each considering several thousand pellets. Furthermore, the pellet viability obtained from both methods suggested an enhanced metabolic activity in glass bead treated pellets during the exponential production phase. However, total viability values differ for flow cytometry (0.32 without and 0.41 with glass beads) and confocal laser scanning microscopy of single stained pellet slices (life ratio in production phase of 0.10 without and 0.22 with glass beads), which is probably caused by the different numbers of investigated pellets. In confocal laser scanning microscopy only one pellet per sample could be investigated while flow cytometry considered at least 50 pellets per sample, resulting in an increased statistical reliability

Understanding and controlling filamentous growth of fungal cell factories: Novel tools and opportunities for targeted morphology engineering

Filamentous fungal cell factories are efficient producers of platform chemicals, proteins, enzymes and natural products. Stirred-tank bioreactors up to a scale of several hundred m³ are commonly used for their cultivation. Fungal hyphae self-assemble into various cellular macromorphologies ranging from dispersed mycelia, loose clumps, to compact pellets. Development of these macromorphologies is so far unpredictable but strongly impacts productivities of fungal bioprocesses. Depending on the strain and the desired product, the morphological forms vary, but no strain- or product-related correlations currently exist to improve process understanding of fungal production systems. However, novel genomic, genetic, metabolic, imaging and modelling tools have recently been established that will provide fundamental new insights into filamentous fungal growth and how it is balanced with product formation. In this primer, these tools will be highlighted and their revolutionary impact on rational morphology engineering and bioprocess control will be discussed.DFG, 315305620, Untersuchung des Einflusses von Scherkräften auf das morphogenetische Gennetzwerk, die Zellintegrität, mikroskopische und makroskopische Morphologie von Aspergillus niger sowie Bildungsraten intra- und extrazellulärer ProdukteDFG, 315384307, Verallgemeinerte morphologische Modellierung aggregierender, filamentöser MikroorganismenDFG, 315457657, Untersuchung und Modellierung der mechanischen und Oberflächen-induzierten Beanspruchung von Pellets filamentöser Mikroorganismen am Beispiel von Lechevalieria aerocolonigenesTU Berlin, Open-Access-Mittel – 202

Modeling the Separation of Microorganisms in Bioprocesses by Flotation

Bioprocesses for the production of renewable energies and materials lack efficient separation processes for the utilized microorganisms such as algae and yeasts. Dissolved air flotation (DAF) and microflotation are promising approaches to overcome this problem. The efficiency of these processes depends on the ability of microorganisms to aggregate with microbubbles in the flotation tank. In this study, different new or adapted aggregation models for microbubbles and microorganisms are compared and investigated for their range of suitability to predict the separation efficiency of microorganisms from fermentation broths. The complexity of the heteroaggregation models range from an algebraic model to a 2D population balance model (PBM) including the formation of clusters containing several bubbles and microorganisms. The effect of bubble and cell size distributions on the flotation efficiency is considered by applying PBMs, as well. To determine the sensitivity of the results on the model assumptions, the modeling approaches are compared, and suggestions for their range of applicability are given. Evaluating the computational fluid dynamics (CFD) of a dissolved air flotation (DAF) system shows the heterogeneity of the fluid dynamics in the flotation tank. Since analysis of the streamlines of the tank show negligible back mixing, the proposed aggregation models are coupled to the CFD data by applying a Lagrangian approach

Inside mycelium - synchrotron radiation and image processing to unveil the time-resolved three-dimensional growth of filamentous fungal pellets

The industrial production of citric acid using the filamentous fungus Aspergillus niger started more than 100 years ago. From then on, industrial biotechnology was born and filamentous fungi began their triumph in many production processes. Nowadays, filamentous fungi produce a diverse range of products, e.g., antibiotics, proteins, acids, or commodities for the pharma, feed, food, fuel, textile, and chemical industries. The efficiency of submerged production processes depends on the developed three-dimensional (3D) hyphal structures. In many industrial processes, fungal pellets – spherical clusters of hyphae – are the preferred morphology. Since pellets cause a low viscosity of the fermentation broth, less energy input is required and a uniform oxygen supply in the culture broth is facilitated. However, substrate limitation could occur in central parts of the pellets and limit productivities. To estimate the diffusion-limited availability of substrates inside pellets, the so far unexplored 3D micro-morphology and the diffusive mass transport through the fibrous network have to be known. Based on X-ray microcomputed tomography (μCT) measurements of freeze-dried pellets and 3D image analysis thereof, we developed the first method to study the 3D location of hyphae, tips, and branches within whole and intact fungal pellets from Aspergillus niger and Penicillium chrysogenum. Further, we computed the effective diffusivity through the pellet micro-morphology gained from the μCT measurements and found a generalized law for the diffusion of substrates through hyphal networks.The morphological heterogeneity of pellets in a fermentation broth makes it necessary to measure a few hundred pellets per cultivation time step to investigate their 3D structural development during pellet growth. At this point, the usage of lab scale µCT has its limitations. A small field of view and long measurement times enable to analyze only a small number of pellets per day (~50, depending on pellet diameter). To overcome this limitation, we applied synchrotron radiation based X-ray microtomography at the P05 beamline (HZG) at PETRAIII (Deutsches Elektronen-Synchrotron - DESY). Due to an optimized sample preparation method and a large field of view, we measured up to 500 fungal pellets from one cultivation time step per scan. With a developed 3D image processing workflow, it is possible to segment the reconstructed volume data into single pellets and to analyze their 3D microscopic inner structures. Based on fast scan times of five minutes, we scanned 31 time steps from 26 hours of fermentation. Thus, the 3D structural development during pellet growth can be tracked time-resolved with a statistically representative number of pellets. Corresponding data will be presented. The synchrotron radiation based X-ray microtomography and the subsequent image processing workflow enable to study and to understand the structural development of fungal pellets during cultivation in highly resolved 3D for the first time. Further, population heterogeneities of a whole fermentation can be analyzed. The gained data will also serve as novel input for morphological modeling approaches

Synchrotron radiation-based X-ray microtomography for three-dimensional growth analysis of Aspergillus niger pellets

Filamentous fungi are an indispensable part of industrial biotechnology. Submerged cultivated in bioreactors with several 100 m³ capacity, these cell factories produce relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to many high-throughput methods to quantify their macromorphology [1]. Nevertheless, only micro-computed tomography (µCT) is the method of choice to study the three-dimensional micromorphology of fungal pellets during submerged cultivation [2]. However, as morphological heterogeneity of pellets makes it necessary to measure hundreds of pellets per sampling time, there is a need for high-throughput µCT approaches.To meet this challenge, we applied synchrotron radiation based X-ray microtomography at the P05 beamline of PETRAIII (Deutsches Elektronen Synchrotron - DESY) and extended our developed method [2], to generate and analyze 3D images of ~20,000 single fungal pellets. We revealed micro-morphological properties such as number and density of spores, tips, branches, and hyphae from 26 sampling points during 48-hour Aspergillus niger cultivations. The computed data allowed us to follow the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time.With our previously developed methods for diffusion computations and growth simulations of filamentous fungal pellets [3][4], the generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth and substrate supply of fungal cultivations.[1] Müller, Barthel, Schmideder et al., Biotechnol. Bioeng., 2022, doi: 10.1002/bit.28124[2] Schmideder, Barthel et al., Biotechnol. Bioeng., 2019, doi: 10.1002/bit.26956[3] Schmideder, Barthel, Müller et al., Biotechnol. Bioeng., 2019, doi: 10.1002/bit.27166[4] Schmideder, Müller, Barthel et al, Biotechnol. Bioeng., 2020, doi: 10.1002/bit.27622

Universal law for diffusive mass transport through mycelial networks

Filamentous fungal cell factories play a pivotal role in biotechnology and circular economy. Hyphal growth and macroscopic morphology are critical for product titers; however, these are difficult to control and predict. Usually pellets, which are dense networks of branched hyphae, are formed during industrial cultivations. They are nutrient‐ and oxygen‐depleted in their core due to limited diffusive mass transport, which compromises productivity of bioprocesses. Here, we demonstrate that a generalized law for diffusive mass transport exists for filamentous fungal pellets. Diffusion computations were conducted based on three‐dimensional X‐ray microtomography measurements of 66 pellets originating from four industrially exploited filamentous fungi and based on 3125 Monte Carlo simulated pellets. Our data show that the diffusion hindrance factor follows a scaling law with respect to the solid hyphal fraction. This law can be harnessed to predict diffusion of nutrients, oxygen, and secreted metabolites in any filamentous pellets and will thus advance the rational design of pellet morphologies on genetic and process levels.DFG, 198187031, Mikro-Computertomograph mit integrierter Materialprüfmaschine und KühleinheitDFG, 315305620, Untersuchung des Einflusses von Scherkräften auf das morphogenetische Gennetzwerk, die Zellintegrität, mikroskopische und makroskopische Morphologie von Aspergillus niger sowie Bildungsraten intra- und extrazellulärer ProdukteDFG, 315384307, Verallgemeinerte morphologische Modellierung aggregierender, filamentöser MikroorganismenDFG, 427889137, Kontrolle von Monospezies und Multispezies Pellet-Heterogenitäten und deren Auswirkungen auf Produktbildung in der Zellfabrik Aspergillus nige