BackgroundFilamentous 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.ResultsWe 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.ConclusionsThis 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.

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Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large-scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production-scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two- and three-compartment scale-down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab-scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale-down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three-compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single-cell size distribution was observed under oscillatory growth conditions with three-dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single-cell-based analysis provides useful information about the homogeneity of responses among the population.

BackgroundThe 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.ResultsAutomatic 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.ConclusionIt 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0922-y) contains supplementary material, which is available to authorized users.