3D imaging and analysis to unveil the impact of microparticles on the pellet morphology of filamentous fungi.

Controlling the morphology of filamentous fungi is crucial to improve the performance of fungal bioprocesses. Microparticle-enhanced cultivation (MPEC) increases productivity, most likely by changing the fungal morphology. However, due to a lack of appropriate methods, the exact impact of the added microparticles on the structural development of fungal pellets is mostly unexplored. In this study synchrotron radiation-based microcomputed tomography and three-dimensional (3D) image analysis were applied to unveil the detailed 3D incorporation of glass microparticles in nondestructed pellets of Aspergillus niger from MPEC. The developed method enabled the 3D analysis based on 375 pellets from various MPEC experiments. The total and locally resolved volume fractions of glass microparticles and hyphae were quantified for the first time. At increasing microparticle concentrations in the culture medium, pellets with lower hyphal fraction were obtained. However, the total volume of incorporated glass microparticles within the pellets did not necessarily increase. Furthermore, larger microparticles were less effective than smaller ones in reducing pellet density. However, the total volume of incorporated glass was larger for large microparticles. In addition, analysis of MPEC pellets from different times of cultivation indicated that spore agglomeration is decisive for the development of MPEC pellets. The developed 3D morphometric analysis method and the presented results will promote the general understanding and further development of MPEC for industrial application.

Selective anomer crystallization from aqueous solution: Monitoring lactose recovery under mutarotation limitation via attenuated total reflection Fourier-transform spectroscopy and theoretical rate analysis.

Lactose is typically produced via cooling crystallization either from whey or whey permeate (edible grade) or from aqueous solution (pharmaceutical grade). While in solution, lactose is present in 2 anomeric forms, α- and ß-lactose. During cooling crystallization under standard process conditions, only α-lactose crystallizes, depleting the solution of α-anomer. In practice, mutarotation kinetics are often assumed to be much faster than crystallization. However, some literature reports limitation of crystallization by mutarotation. In the present research, we investigate the influence of operating conditions on mutarotation in lactose crystallization and explore the existence of an operation regimen where mutarotation can be disregarded in the crystallization process. Therefore, we study crystallization from aqueous lactose solutions by inline monitoring of concentrations of α- and ß-lactose via attenuated total reflection Fourier-transform spectroscopy. By implementing a linear cooling profile of 9 K/h to a minimum temperature of 10°C, we measured a remarkable increase in ß/α ratio, reaching a maximum of 2.19. This ratio exceeds the equilibrium level by 36%. However, when the same cooling profile was applied to a minimum temperature of 25°C, the deviation was significantly lower, with a maximum ß/α ratio of 1.72, representing only an 8% deviation from equilibrium. We also performed a theoretical assessment of the influence of process parameters on crystallization kinetics. We conclude that mutarotation needs to be taken into consideration for efficient crystallization control if the crystal surface area and supersaturation are sufficiently high.

Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials.

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air-liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air-liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets.

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron [German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 h Aspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.

From spores to fungal pellets: A new high-throughput image analysis highlights the structural development of Aspergillus niger.

Many filamentous fungi are exploited as cell factories in biotechnology. Cultivated under industrially relevant submerged conditions, filamentous fungi can adopt different macromorphologies ranging from dispersed mycelia over loose clumps to pellets. Central to the development of a pellet morphology is the agglomeration of spores after inoculation followed by spore germination and outgrowth into a pellet population, which is usually very heterogeneous. As the dynamics underlying population heterogeneity is not yet fully understood, we present here a new high-throughput image analysis pipeline based on stereomicroscopy to comprehensively assess the developmental program starting from germination up to pellet formation. To demonstrate the potential of this pipeline, we used data from 44 sampling times harvested during a 48 h submerged batch cultivation of the fungal cell factory Aspergillus niger. The analysis of up to 1700 spore agglomerates and 1500 pellets per sampling time allowed the precise tracking of the morphological development of the overall culture. The data gained were used to calculate size distributions and area fractions of spores, spore agglomerates, spore agglomerates within pellets, pellets, and dispersed mycelia. This approach eventually enables the quantification of culture heterogeneities and pellet breakage.

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.

Capturing Crystal Shape Evolution from Molecular Simulations.

A simple and efficient algorithm for tracking shape evolution of small-molecule organic crystals during molecular simulations is described. It is based on the reconstruction of a crystal surface from molecular coordinates using an alpha-shape triangulation algorithm followed by the DBSCAN clustering of neighboring triangles with similar normal vectors to crystal faces. No information except the unit cell parameters is needed beforehand, enabling the user to automatically detect not only existing but also new forming crystal faces and edges, which is valuable for prediction of growth and dissolution kinetics. The results are demonstrated for aspirin and paracetamol crystals.

Swelling properties of roasted coffee particles.

BACKGROUND: In this study, the swelling behavior of roasted coffee particles in water and particularly its impact on particle diameter is examined by applying laser-diffraction analysis and microscopy. Several potential influencing factors are investigated: initial particle size, roasting degree, and temperature. Additionally, the time dependency of swelling and particle shape is evaluated at two different temperatures. RESULTS: We verify that particle erosion occurs - as observed by an increase of the fine particle fraction after wetting - and it is revealed that this effect is more pronounced with a rise in temperature. The total relative increase in particle size is determined as approximately 15% based on a broad range of different sized coffee grounds. It is demonstrated that the degree of swelling is independent of both the initial particle diameter and the roasting degree. The particle shape is found to be unaffected by swelling. This research reveals that swelling is initially quick, with 60-80% of the final steady-state diameter being reached after 30 s and completed after 4 min of wetting, i.e. within the timescale of conventional coffee brewing methods. CONCLUSION: This work provides a better understanding of the impact of wetting as part of the coffee brewing process, thus aiding the design, modeling, and optimization of coffee extraction. It clarifies the strong deviation of previous results on coffee-particle swelling by considering particle erosion and degassing and provides a robust method for quantification. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

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

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. Three-dimensional 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 three-dimensional 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.

Temperature profile optimization: potential for multi-enzymatic biopolymer depolymerization processes.

Optimal control of temperature was applied to a population balance model of enzymatically catalyzed depolymerization of a soluble polymer coupled with denaturation of enzyme. The reaction time required to reach a desired yield was predicted to be reduced by more than 10[Formula: see text] compared with isothermal operation. Also the yield within a given time could be increased by more than 5[Formula: see text] points. It was also possible to increase the yield and reduce the reaction time if a time-varying temperature profile was used. Furthermore, a simple-to-implement linear increasing temperature profile was shown to realize most of the saving potential. Rigorous optimization of the enzyme mixture and composition was predicted to have an even greater potential for improving the economic feasibility of the process. Optimization coupled with optimal control can be performed quickly in silico using the algorithm developed in this study if a validated and parameterized population balance model is available.

Estimating Colloidal Contact Model Parameters Using Quasi-Static Compression Simulations.

For colloidal particles interacting in suspensions, clusters, or gels, contact models should attempt to include all physical phenomena experimentally observed. One critical point when formulating a contact model is to ensure that the interaction parameters can be easily obtained from experiments. Experimental determinations of contact parameters for particles either are based on bulk measurements for simulations on the macroscopic scale or require elaborate setups for obtaining tangential parameters such as using atomic force microscopy. However, on the colloidal scale, a simple method is required to obtain all interaction parameters simultaneously. This work demonstrates that quasi-static compression of a fractal-like particle network provides all the necessary information to obtain particle interaction parameters using a simple spring-based contact model. These springs provide resistances against all degrees of freedom associated with two-particle interactions, and include critical forces or moments where such springs break, indicating a bond-breakage event. A position-based cost function is introduced to show the identifiability of the two-particle contact parameters, and a discrete, nonlinear, and non-gradient-based global optimization method (simplex with simulated annealing, SIMPSA) is used to minimize the cost function calculated from deviations of particle positions. Results show that, in principle, all necessary contact parameters for an arbitrary particle network can be identified, although numerical efficiency as well as experimental noise must be addressed when applying this method. Such an approach lays the groundwork for identifying particle-contact parameters from a position-based particle analysis for a colloidal system using just one experiment. Spring constants also directly influence the time step of the discrete-element method, and a detailed knowledge of all necessary interaction parameters will help to improve the efficiency of colloidal particle simulations.

Insights into pharmaceutical nanocrystal dissolution: a molecular dynamics simulation study on aspirin.

The presented molecular dynamics simulations are the first simulations to reveal dynamic dissolution of a pharmaceutical crystal in its experimentally determined shape. Continuous dissolution at constant undersaturation of the surrounding medium is ensured by introducing a plane of sticky dummy atoms into the water slab. These atoms have a strong interaction potential with dissolved aspirin molecules, but interactions with water are excluded from the calculations. Thus, the number of aspirin molecules diffusing freely in solution is kept at a low value and continuous dissolution of the aspirin crystal is monitored. Further insight into face-specific dissolution is drawn. The dissolution mechanism of receding edges is found for the (001) plane. These findings are in good agreement with experimental results. While the proposed dissolution mechanism for the (100) plane is terrace sinking on a rough surface, no pronounced dissolution of the perfectly flat face is seen in the present work. Molecular simulations of pharmaceuticals in their experimentally obtained structure therefore have shown to be especially suited for the investigation of dissolving faces, where the edges have a pronounced effect. In contrast to previous studies a propagation of the dissolution front into the crystal face is reported, and the crystal bulk is stable over the whole simulation time of 150 ns.

Breaking down barriers: comprehensive functional analysis of the Aspergillus niger chitin synthase repertoire.

BACKGROUND: Members of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom. RESULTS: In this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization. CONCLUSION: This study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.

Predicting the essential oil composition in supercritical carbon dioxide extracts from hop pellets using mathematical modeling.

Supercritical fluid extraction from hops (Humulus lupulus L.) can be used to extract essential oil for the flavoring of beer. With a special focus on the oil composition being linked to the hop aroma, the influence of pressure and temperature on the extraction kinetics of seven oil components (ß-myrcene, α-humulene, ß-caryophyllene, 2-methylbutyl isobutyrate, undecanone, linalool, and α-pinene) is analyzed and modeled in this article. Supercritical CO2 extraction from hop pellets was conducted at pressure-temperature combinations of 90/100/110 bar and 40/45/50 °C. The extract composition over time, analyzed by gas chromatography, was used for the parameterization of two existing mechanistic models: an internal-mass-transfer-control (IMTC), and a broken-and-intact-cells (BIC) model. The IMTC model was found to effectively describe most extraction kinetics and hence applied in this study. In contrast to previous studies, the IMTC model parameters were not only fitted to individual extraction curves from different experiments but also correlated to temperature and pressure as a further step towards model-based prediction. Using the parameterized model, the extract composition was predicted at 95 bar/48 °C, 105 bar/42 °C, and 105 bar/48 °C. Extraction yields were found to be higher at lower temperatures and higher pressures in general. The sensitivity towards pressure was observed to differ between components and to be particularly higher for ß-myrcene compared with α-humulene. Changes of the essential oil composition with a variation in pressure and temperature were predicted correctly by the model with a mean relative deviation from experimental data of 11.7% (min. 1.2%, max. 36.2%).

Influence of Flow Rate, Particle Size, and Temperature on Espresso Extraction Kinetics.

Brewing espresso coffee (EC) is considered a craft and, by some, even an art. Therefore, in this study, we systematically investigated the influence of coffee grinding, water flow rate, and temperature on the extraction kinetics of representative EC components, employing a central composite experimental design. The extraction kinetics of trigonelline, caffeine, 5-caffeoylquinic acid (5-CQA), and Total Dissolved Solids (TDS) were determined by collecting and analyzing ten consecutive fractions during the EC brewing process. From the extraction kinetics, the component masses in the cup were calculated for Ristretto, Espresso, and Espresso Lungo. The analysis of the studied parameters revealed that flow rate had the strongest effect on the component mass in the cup. The intensity of the flow rate influence was more pronounced at finer grindings and higher water temperatures. Overall, the observed influences were minor compared to changes resulting from differences in total extracted EC mass.

Investigating the Role of Odorant-Polymer Interactions in the Aroma Perception of Red Wine: A Density Functional Theory-Based Approach.

The aroma of red wine results from the intricate interplay between aroma compounds (odorants) and complex polymers generated during fermentation. This study combines density functional theory (DFT), human sensory experiments, and nuclear magnetic resonance to investigate the impact of odorant-polymer interactions on wine aroma. Molecular aggregation patterns of odorants with polymer segments are identified, indicating the crucial role of intermolecular noncovalent interactions, such as hydrogen bonds and van der Waals interactions, in stabilizing odorant-polymer conformations. Certain odorants, including 3-isobutyl-2-methoxypyrazine and cis-whisky lactone, exhibit high binding affinity to specific polymer segments, such as (+)-catechin and p-coumaric acid, resulting in substantial changes in the perceived aroma. Their strong binding affinities correlate with changes in sensory experiments for binary mixtures. The results provide insights into the molecular mechanisms of odorant-polymer interactions in red wine with the potential of DFT calculations as a tool for predicting and tailoring red wine aroma.

Mechanical, physical and thermal properties of composite materials produced with the basidiomycete Fomes fomentarius.

BACKGROUND: To achieve climate neutrality, fundamentally new concepts of circularity need to be implemented by the building sector as it contributes to 40% of anthropogenic CO2 emission. Fungal biotechnology can make a significant contribution here and help eliminate fossil dependency for building material production. Recently, we have shown that the medicinal polypore Fomes fomentarius feeds well on renewable lignocellulosic biomass and produces composite materials that could potentially replace fossil fuel-based expanded polystyrene as insulation material. RESULTS: In this study, we explored the mechanical, physical, and thermal properties of F. fomentarius-based composite materials in more detail and determined key performance parameters that are important to evaluate the usability of F. fomentarius-based composite materials in the construction sector. These parameters were determined according to European standards and included compressive strength, modulus of elasticity, thermal conductivity, water vapour permeability, and flammability of uncompressed composites as well as flexural strength, transverse tensile strength, and water absorption capacity of heat-pressed composites, among others. We could show that uncompressed composites obtained from F. fomentarius and hemp shives display a thermal conductivity of 0.044 W (m K)-1 which is in the range of natural organic fibres. A water vapour permeability of 1.72 and classification into flammability class B1 clearly surpasses fossil-based insulation materials including expanded polystyrene and polyurethane. We could furthermore show that heat-pressing can be used to reliably generate stiff and firm particleboards that have the potential to replace current wood-based particleboards that contain synthetic additives. X-ray microcomputed tomography finally visualized for the first time the growth of hyphae of F. fomentarius on and into the hemp shive substrates and generated high-resolution images of the microstructure of F. fomentarius-based composites. CONCLUSION: This study demonstrates that fungal-based composites produced with F. fomentarius partially meet or even exceed key performance parameters of currently used fossil fuel-based insulation materials and can also be used to replace particleboards.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Restructuring of colloidal aggregates in shear flow: coupling interparticle contact models with Stokesian dynamics.

A method to couple interparticle contact models with Stokesian dynamics (SD) is introduced to simulate colloidal aggregates under flow conditions. The contact model mimics both the elastic and plastic behavior of the cohesive connections between particles within clusters. Owing to this, clusters can maintain their structures under low stress while restructuring or even breakage may occur under sufficiently high stress conditions. SD is an efficient method to deal with the long-ranged and many-body nature of hydrodynamic interactions for low Reynolds number flows. By using such a coupled model, the restructuring of colloidal aggregates under shear flows with stepwise increasing shear rates was studied. Irreversible compaction occurs due to the increase of hydrodynamic stress on clusters. Results show that the greater part of the fractal clusters are compacted to rod-shaped packed structures, while the others show isotropic compaction.