Abstract
BackgroundFilamentously growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. However, limited experimental throughput in bioprocess development still hampers taking advantage of their full potential. Miniaturization and automation are powerful tools to accelerate bioprocess development, but so far the application of such technologies has mainly been focused on non-filamentous systems. During cultivation, filamentous fungi can undergo remarkable morphological changes, creating challenging cultivation conditions. Depending on the process and product, only one specific state of morphology may be advantageous to achieve e.g. optimal productivity or yield. Different approaches to control morphology have been investigated, such as microparticle enhanced cultivation. However, the addition of solid microparticles impedes the optical measurements typically used by microbioreactor systems and thus alternatives are needed.ResultsAspergillus giganteus IfGB 0902 was used as a model system to develop a time-efficient and robust workflow allowing microscale cultivation with increased throughput. The effect of microtiter plate geometry, shaking frequency and medium additives (talc and calcium chloride) on homogeneity of culture morphology as well as reproducibility were analyzed via online biomass measurement, microscopic imaging and cell dry weight. While addition of talc severely affected online measurements, 2% (w v−1) calcium chloride was successfully applied to obtain a highly reproducible growth behavior with homogenous morphology. Furthermore, the influence of small amounts of complex components was investigated for the applied model strain. By correlation to cell dry weight, it could be shown that optical measurements are a suitable signal for biomass concentration. However, each correlation is only applicable for a specific set of cultivation parameters. These optimized conditions were used in micro as well as lab-scale bioreactor cultivation in order to verify the reproducibility and scalability of the setup.ConclusionA robust workflow for A. giganteus was developed, allowing for reproducible microscale cultivation with online monitoring, where calcium chloride is an useful alternative to microparticle enhanced cultivation in order to control the morphology. Independent of the cultivation volume, comparable phenotypes were observed in microtiter plates and in lab-scale bioreactor.
Highlights
Growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology
Filamentous microorganisms have always played an important role in industrial biotechnology: Citric acid production with Aspergillus niger dates back to 1919 in Belgium [1] and since Aspergillus sp. have been established as industrial workhorses
Microbioreactor reference cultivation As a starting point, standard conditions for bacterial microbioreactor cultivation were applied to Aspergillus giganteus in the BioLector setup
Summary
Growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. 27% of commercial enzyme production, such as amylases, catalases and proteases, is performed utilizing Aspergillus [2]. Since a steadily increasing number of genomes become available, an increase in their utilization as a source for novel enzymes and metabolites is expected [4] Due to their complex morphology, the development of industrial processes using filamentous fungi remains quite challenging. The mycelia can either be freely dispersed throughout the liquid volume or form macroscopic aggregates If such aggregates form rather loose structures, they are referred to as mycelial clumps, whereas more compact and dense structures are called pellets [6]. The suitable morphology has to be identified for each strain and process individually
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