Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary metabolites

Background Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. Results We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) and a BioFlo STR (New Brunswick®), respectively. Two different A. niger strains were analysed, which produce heterologously the commercial drug enniatin B. Both strains expressed the esyn1 gene that encodes a non-ribosomal peptide synthetase ESYN under control of the inducible Tet-on system, but differed in their dependence on feeding with the precursors d-2-hydroxyvaleric acid and l-valine. Cultivations of A. niger in the CELL-tainer resulted in the formation of large pellets, which were heterogeneous in size (diameter 300–800 μm) and not observed during STR cultivations. When talcum microparticles were added, it was possible to obtain a reduced pellet size and to control pellet heterogeneity (diameter 50–150 μm). No foam formation was observed under wave-mixed cultivation conditions, which made the addition of antifoam agents needless. Overall, enniatin B titres of about 1.5–2.3 g L−1 were achieved in the CELL-tainer® system, which is about 30–50% of the titres achieved under STR conditions. Conclusions This is the first report studying the potential use of single-use wave-mixed reactor systems for the cultivation of A. niger. Although final enniatin yields are not competitive yet with titres achieved under STR conditions, wave-mixed cultivations open up new avenues for the cultivation of shear-sensitive mutant strains as well as high cell-density cultivations.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli

Microbial cultivation in rocking single-use bioreactors

The application of single-use bioreactors (SUB) for microbial cultivation, especially of reactor designs beyond the traditional stirred tank, is usually regarded as crucial. Especially the usually low gas mass transfer coefficients are insufficient, however this is not true for 2-dimensionally rocking motion bioreactors like the CELL-tainer®. Volumetric gas mass transfer coefficients (kLa-values) of 600 h-1 are achieved (fig. 1), which allow bacterial fed-batch cultivations up to a cell density of 50 gL-1 at growth rates of 0.3 h-1 w/o any oxygen blending (Junne et al, Chem Eng Technol 2013, 85, p. 57-66). One major asset in this respect are the low maximum shear forces in comparison to stirred tank reactors. This feature might be beneficial when shear sensitive microbes are cultivated, like marine phototrophic and heterotrophic microalgae and filamentous organisms. Please click Additional Files below to see the full abstract

Monitoring der Einzelzellmorphologie zur Bewertung mikrobieller eukaryotischer Bioprozesse

Cell morphology is not only influenced by the cell cycle, the aging or individual properties, but also by environmental impacts such as those occurring on a large-scale. Cell morphology can be a suitable parameter for in situ measurements as it changes dynamically and is often related to cell physiology. In order to be able to identify relationships between cell physiology and morphology, statistically representative amounts of data have to be measured. It should be considered that the behaviour of cells is not only dynamic, but also very sensitive to environmental changes. Therefore, off line measurements may not be suitable for detecting small changes in the morphological properties. Sampling and sample preparation would conceal this, apart from an often insufficient number of data or an unreasonable amount of time and effort. Among the techniques that are able to capture the morphological characteristics of cells in real-time, automated imaging technologies are promising, because they provide additional information about cellular structures, shape and cell aggregation beyond size. Photo-optical in situ microscopy (ISM) and three-dimensional holographic microscopy (DHM) were used in this study to measure the morphological dynamics in eukaryotic cultures on a single-cell basis, using heterotrophic algae and yeast as examples. The intracellular concentration of the polyunsaturated fatty acid docosahexaenoic acid (DHA) in the heterotrophic algae Cryptecodinium cohnii was monitored. A second order correlation between the DHA content as measured off line chromatographically and the prediction using the average Sauter diameter was found. A different media composition did not only influence the cell size, but also the circularity and phase homogeneity of the algae cells. Consequently, different chloride ion substitutes were tested with respect to the cell growth and lipid accumulation in C. cohnii. Multi-compartment reactors were used to investigate the influence of gradients, as they occur in a large-scale, on the morphological heterogeneity in Saccharomyces cerevisiae cultures. Contrary to expectations, the sterol synthesis was positively influenced by oscillatory oxygen availability (ergosterol ester concentrations increased by 75 %), although microbial growth was decreased (the biomass concentration was reduced by 20 %). Budding of yeast was monitored in batch cultivation using ISM. A narrow size distribution was measured during the growth phase while the population homogeneity increased. If glucose was depleted, the percentage of non-budding cells remained almost constant due to a significant reduction in growth activity. The ratio of budding and total cells was successfully applied to differentiate between the different growth stages. The methods proved to be suitable for monitoring morphological properties over a relevant concentration range. Faster particle identification, including overlapping particles, and further investigations to better understand the relations between the shape and state of a cell will allow the technology to be used to control a variety of bioprocesses.Die Zellmorphologie wird nicht nur durch den Zellzyklus, die Alterungs- oder individuelle Eigenschaften beeinflusst, sondern auch durch Umweltbelastungen, wie sie z.B. im großen Maßstab auftreten. Die Zellmorphologie kann ein geeignete Parameter für eine in situ Messung sein, da sie sich dynamisch verändert und dabei oft mit der Zellphysiologie zusammenhängt. Um Beziehungen zwischen der Zellphysiologie und Morphologie identifizieren zu können, müssen statistisch repräsentative Datenmengen gemessen werden. Zu Berücksichtigen ist, dass das Verhalten der Zellen dabei nicht nur extrem dynamisch ist, sondern auch sehr sensibel gegenüber Umweltveränderungen. Daher können off line Messungen ungeeignet sein, kleinere Änderungen in den morphologischen Eigenschaften zu detektieren. Die Probenahme und in der Regel die Probenvorbereitung würde diese überdecken, abgesehen von einer oft nicht ausreichenden Anzahl von Daten oder eines nicht vertretbaren Aufwandes. Unter den Techniken, die in der Lage sind, morphologische Merkmale von Zellen zeitnah zu erfassen, sind automatisierte Bildgebungstechnologien vielversprechend, da sie über die Größe hinaus weitere Informationen über zelluläre Strukturen, Form und Zellaggregation liefern. Die photo-optische in situ Mikroskopie (ISM) und die drei-dimensionale holographische Mikroskopie (DHM) wurden in dieser Studie zur Messung der morphologischen Dynamik in eukaryontischen Kulturen auf Einzelzellbasis eingesetzt, die exemplarisch an heterotrophen Algen und Hefe untersucht wurde. Die intrazelluläre Konzentration der mehrfach ungesättigten Fettsäure Docosahexaensäure (DHA) in der heterotrophen Alge Cryptecodinium cohnii wurde überwacht. Eine Korrelation zweiter Ordnung zwischen dem DHA-Gehalt, wie er off line chromatographisch gemessen wurde, und der Vorhersage unter Verwendung des Durchschnitts des Sauter-Durchmessers konnte gefunden werden. Eine unterschiedliche Medienzusammensetzung beeinflusste nicht nur die Zellgröße, sondern auch die Zirkularität und Phasenhomogenität der Algenzellen. Folglich wurden verschiedene Chloridionenersatzstoffe hinsichtlich des Zellwachstums und der Lipidakkumulation in C. cohnii getestet. Mehrkompartimenten-Reaktoren wurden eingesetzt, um den Einfluss von Gradienten, wie sie im großen Maßstab auftreten, auf die morphologische Heterogenität innerhalb von Saccharomyces cerevisiae Kulturen zu untersuchen. Insbesondere wurden die Auswirkungen von Sauerstofflimitierungen auf die Zellheterogenität untersucht. Entgegen den Erwartungen wurde die Sterolsynthese durch die oszillatorische Sauerstoffverfügbarkeit positiv beeinflusst (die Ergosterolesterkonzentrationen stiegen um 75 %), obwohl sich das mikrobielle Wachstum verlangsamte (die Biomassekonzentration war um 20 % erniedrigt). Darüber hinaus wurde die Knospung der Hefe mit Hilfe der ISM auf Einzelzellebene in Batchkultivierungen überwacht. Die Größenverteilung wurde während der Wachstumsphase enger, so dass die Populationshomogenität zunahm. War die Glukose verbraucht, blieb der Prozentsatz der Nicht-knospenden Zellen aufgrund einer stark verminderten Wachstumsaktivität nahezu konstant. Anhand des Anteils knospender Zellen konnte zwischen den verschiedenen Kultivierungsstadien unterschieden werden. Die gezeigten Methoden erwiesen sich als geeignet zur Überwachung morphologischer Eigenschaften über einen relevanten Konzentrationsbereich hinweg. Eine schnellere Partikelidentifizierung, auch von überlappenden Partikeln, und weitere Untersuchungen zu einem besseren Verständnis der Zusammenhänge zwischen Form und Zustand einer Zelle wird den Einsatz der Technologie zur Kontrolle einer Vielzahl an Bioprozessen erlauben.BMEL, 511-06.01-28-1-73.026-10, FENA − fishmeal and oil feed substitutes for a sustainable aquacultureBMWi, ZF 4184201CR5, Smart Process Inspectio

Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy

Abstract Background The morphology of yeast cells changes during budding, depending on the growth rate and cultivation conditions. A photo-optical microscope was adapted and used to observe such morphological changes of individual cells directly in the cell suspension. In order to obtain statistically representative samples of the population without the influence of sampling, in situ microscopy (ISM) was applied in the different phases of a Saccharomyces cerevisiae batch cultivation. The real-time measurement was performed by coupling a photo-optical probe to an automated image analysis based on a neural network approach. Results Automatic cell recognition and classification of budding and non-budding cells was conducted successfully. Deviations between automated and manual counting were considerably low. A differentiation of growth activity across all process stages of a batch cultivation in complex media became feasible. An increased homogeneity among the population during the growth phase was well observable. At growth retardation, the portion of smaller cells increased due to a reduced bud formation. The maturation state of the cells was monitored by determining the budding index as a ratio between the number of cells, which were detected with buds and the total number of cells. A linear correlation between the budding index as monitored with ISM and the growth rate was found. Conclusion It is shown that ISM is a meaningful analytical tool, as the budding index can provide valuable information about the growth activity of a yeast cell, e.g. in seed breeding or during any other cultivation process. The determination of the single-cell size and shape distributions provided information on the morphological heterogeneity among the populations. The ability to track changes in cell morphology directly on line enables new perspectives for monitoring and control, both in process development and on a production scale