Members of the genus Streptomyces are recognized by their remarkable biosynthetic potential. It has been reported that the culture morphology strongly correlates with secondary metabolite production. In this work, we have established a method for sample preparation for microscopy from very complex fermentation broth samples, using crystal violet staining. Further, we developed a process of digital image analysis and applied it for morphological analysis of the clavulanic acid - producing Streptomyces clavuligerus. Our results show, that pelleted growth and differentiation processes can be observed during growth and development of S. clavuligerus cultures at specific conditions in shake flask cultures. Interestingly, spore-like structures of the S. clavuligerus culture have been observed in liquid media towards the end of bioprocess at the pilot scale, thus suggesting that the development of Streptomyces culture in liquid media is more similar to the development cycle on solid media. Further on, we analysed different morphological parameters such as length, thickness, branching and mycelium viability and compared the morphology at the pilot and industrial scale. The S. clavuligerus culture in the standard pilot bioreactor formed significantly shorter hyphae in comparison to the pilot bioreactor, which is by its geometry and impeller configuration similar to the industrial vessels. The S. clavuligerus culture grown at the industrial scale formed even shorter hyphae, less branched, but significantly thicker, and more viable compared to the cultures grown in bioreactors at the pilot scale. Our results indicate, there is a correlation between morphology and clavulanic acid production. However, more data is needed to reliably confirm this correlation. We have demonstrated that the methodology developed in this work is very useful for Streptomyces morphology observation and evaluation and it is suitable for routine use in the industrial environment.
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