Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium

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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0956-1) contains supplementary material, which is available to authorized users.

Revealing nutritional requirements of MICP-relevant Sporosarcina pasteurii DSM33 for growth improvement in chemically defined and complex media

“…The chemically defined medium used in this study is based on an adaption of Poolman medium 45 by Müller et al 27 and adapted again in multiple iteration steps for our purposes (see Supplementary Table S12 online). All ingredients are listed in Table 1 .…”

“…All ingredients are listed in Table 1 . Components are grouped based on Müller et al 27 . The chemically defined medium consists of multiple stock solutions; the L-glutamic acid, L-methionine, L-serine and vitamin stock solution were freshly made for each cultivation.…”

“…Contrary to complex media, growth limitations due to auxotrophic deficiencies and nutritional requirements of an organism can be identified very easily when using chemically defined media 25 – 27 . Knowledge about nutritional requirements and auxotrophic deficiencies can be applied to improve cultivation performance 23 .…”

Metabolomics Analysis Reveals Global Metabolic Changes in the Evolved E. coli Strain with Improved Growth and 1-Butanol Production in Minimal Medium

“…In the fermentation industry, the use of complex media poses several major disadvantages such as being prone to significant lot-to-lot variations, which results in inconsistent and unstable fermentation performance [8,9]. Thus, the use of chemically defined or minimal media is more applicable for commercial fermentation as it ensures consistency among fermentation batches and, hence, would allow for efficient quality control.…”

“…Thus, the use of chemically defined or minimal media is more applicable for commercial fermentation as it ensures consistency among fermentation batches and, hence, would allow for efficient quality control. Furthermore, minimal media are less sensitive to sterilization conditions and permit a more simplified downstream processing [8,9]. For these reasons, improvement in the performance of the 1-butanol-producing E. coli strain in minimal media is required.…”