Abstract
BackgroundCulture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097.ResultsBacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth.ConclusionsThe introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.
Highlights
Culture media containing complex compounds like yeast extract or peptone show numerous disadvantages
Bacillus pumilus DSM 18097 was cultivated in V3 mineral medium [45] that was supplemented with different concentrations of yeast extract (Fig. 2a), peptone (Fig. 2b) and in modified Poolman medium [8] (Fig. 2c)
This study demonstrated that B. pumilus DSM 18097 was able to grow in a simple minimal medium when the strain’s auxotrophies were appropriately identified and the respective nutrients were added to the medium
Summary
Culture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The use of chemically defined media receives more and more attention in commercial fermentations This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. The majority of fastidious microorganisms require miscellaneous trace elements, vitamins and amino acids, which are usually not included in standard mineral media. For this reason, chemically undefined media containing complex components of natural origin are often applied [1, 2]. Yeast extract is one of the most frequently used complex supplements It contains a mixture of carbohydrates, amino acids, peptides, vitamins, trace elements and various other oligomeric compounds [3]. A consistent product quality is extremely difficult to ensure [6]
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