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      Novel bioreactor internals for the cultivation of spore‐forming fungi in pellet form

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          Abstract

          This study introduced an automated long‐term fermentation process for fungals grown in pellet form. The goal was to reduce the overgrowth of bioreactor internals and sensors while better rheological properties in the fermentation broth, such as oxygen transfer and mixing time, can be achieved. Because this could not be accomplished with continuous culture and fed‐batch fermentation, repeated‐batch fermentation was implemented with the help of additional bioreactor internals (“sporulation supports”). This should capture some biomass during fermentation. After harvesting the suspended biomass, intermediate cleaning was performed using a cleaning device. The biomass retained on the sporulation support went through the sporulation phase. The spores were subsequently used as inocula for the next batch. The reason for this approach was that the retained pellets could otherwise cause problems ( e.g., overgrowth on sensors) in subsequent batches because the fungus would then show undesirable hyphal growth. Various sporulation supports were tested for sufficient biomass fixation to start the next batch. A reproducible spore concentration within the range of the requirements could be achieved by adjusting the sporulation support (design and construction material), and an intermediate cleaning adapted to this.

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          Characterization and control of fungal morphology for improved production performance in biotechnology.

          Filamentous fungi have been widely applied in industrial biotechnology for many decades. In submerged culture processes, they typically exhibit a complex morphological life cycle that is related to production performance--a link that is of high interest for process optimization. The fungal forms can vary from dense spherical pellets to viscous mycelia. The resulting morphology has been shown to be influenced strongly by process parameters, including power input through stirring and aeration, mass transfer characteristics, pH value, osmolality and the presence of solid micro-particles. The surface properties of fungal spores and hyphae also play a role. Due to their high industrial relevance, the past years have seen a substantial development of tools and techniques to characterize the growth of fungi and obtain quantitative estimates on their morphological properties. Based on the novel insights available from such studies, more recent studies have been aimed at the precise control of morphology, i.e., morphology engineering, to produce superior bio-processes with filamentous fungi. Copyright © 2012 Elsevier B.V. All rights reserved.
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            The filamentous fungal pellet and forces driving its formation

            Filamentous fungi play an important role not only in the bio-manufacturing of value-added products, but also in bioenergy and environmental research. The bioprocess manipulation of filamentous fungi is more difficult than that of other microbial species because of their different pellet morphologies and the presence of tangled mycelia under different cultivation conditions. Fungal pellets, which have the advantages of harvest ease, low fermentation broth viscosity and high yield of some proteins, have been used for a long time. Many attempts have been made to establish the relationship between pellet and product yield using quantitative approaches. Fungal pellet formation is attributed to the combination of electrostatic interactions, hydrophobicity and specific interactions from spore wall components. Electrostatic interactions result from van der Waals forces and negative charge repulsion from carboxyl groups in the spore wall structure. Electrostatic interactions are also affected by counter-ions (cations) and the physiologic conditions of spores that modify the carboxyl groups. Fungal aggregates are promoted by the hydrophobicity generated by hydrophobins, which form a hydrophobic coat that covers the spore. The specific interactions of spore wall components contribute to spore aggregation through salt bridging. A model of spore aggregation was proposed based on these forces. Additionally, some challenges were addressed, including the limitations of research techniques, the quantitative determination of forces and the complex information of biological systems, to clarify the mechanism of fungal pellet formation.
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              Morphology engineering of Aspergillus niger for improved enzyme production.

              Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher-value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision-induced disruption of conidia aggregates and probably also the hindrance of new spore-spore interactions in the very early stage of the process. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Linked to the formation of freely dispersed mycelium, titers for glucoamylase (GA) expressed as intracellular enzyme (88 U/mL) and fructofuranosidase secreted into the supernatant (77 U/mL), were up to fourfold higher in shake flasks. Moreover, accumulation of the undesired by-product oxalate was suppressed by up to 90%. The microparticle strategy could be successfully transferred to fructofuranosidase production in bioreactor, where a final titer of 160 U/mL could be reached. Using co-expression of GA with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production. (c) 2009 Wiley Periodicals, Inc.
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                Author and article information

                Contributors
                w.soerjawinata@umwelt-campus.de
                Journal
                Eng Life Sci
                Eng Life Sci
                10.1002/(ISSN)1618-2863
                ELSC
                Engineering in Life Sciences
                John Wiley and Sons Inc. (Hoboken )
                1618-0240
                1618-2863
                18 May 2022
                July 2022
                : 22
                : 7 ( doiID: 10.1002/elsc.v22.7 )
                : 474-483
                Affiliations
                [ 1 ] Institute for Biotechnical Process Design Trier University of Applied Sciences, Environmental Campus Birkenfeld Hoppstädten‐Weiersbach Germany
                [ 2 ] Institut für Biotechnologie und Wirkstoff‐Forschung gGmbH (IBWF) Mainz Germany
                [ 3 ] Institute of Pharmaceutical and Biomedical Sciences Johannes Gutenberg University of Mainz Mainz Germany
                [ 4 ] Institute of Bioprocess Engineering Technical University Kaiserslautern Kaiserslautern Germany
                Author notes
                [*] [* ] Correspondence

                Winda Soerjawinata, Institute for Biotechnical Process Design, Trier University of Applied Sciences, Environmental Campus Birkenfeld, Campusallee 9913, 55768 Hoppstädten‐Weiersbach, Germany.

                Email: w.soerjawinata@ 123456umwelt-campus.de

                Author information
                https://orcid.org/0000-0002-7674-0967
                Article
                ELSC1488
                10.1002/elsc.202100094
                9288991
                35865648
                f06bed0d-c966-4cc8-b7ad-8c82bc38803b
                © 2022 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 07 February 2022
                : 28 July 2021
                : 10 February 2022
                Page count
                Figures: 6, Tables: 1, Pages: 10, Words: 6766
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                July 2022
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.7 mode:remove_FC converted:17.07.2022

                bioreactor internals,fungal growth,fungal pellets,repeated‐batch fermentation,sporulation conditions

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