Imaging high-performance thin-layer chromatography as powerful tool to visualize metabolite profiles of eight Bacillus candidates upon cultivation and growth behavior

Abstract

Imaging high-performance thin-layer chromatography (HPTLC) was explored with regard to its ability to visualize changes in the metabolite profile of bacteria. Bacillus subtilis has become a model organism in many fields. The increasing interest in these bacteria is driven by their attributed probiotic activity. However, growth behavior and metabolism of Bacillus species have a considerable influence on their activity and secondary metabolite profile. On the HPTLC plate, cultivation broths of Bacillus species (B. subtilis, B. licheniformis, B. pumilus and B. amyloliquefaciens) and some B. subtilis strains of high genetic similarity up to 99.5% were applied directly and compared with their respective liquid-liquid extracts. The latter as well as the cultivation in a minimal medium reduced the matrix load and improved the zone resolution. Cultivation parameters such as nutrient supply, cultivation temperature, cultivation time and rotational speed (oxygen level) as well as medium change were shown to have a considerable influence on the growth behavior and resulting metabolite profiles. Imaging HPTLC turned out to be an efficient and affordable tool to visualize such influences of cultivation parameters on the metabolite profiles. It converts the complexity of reaction processes occurring during cell cultivation in easy-to-understand images, which are helpful to figure out factors of influence and understand activity changes. The results highlighted that optimal cultivation conditions need to be found for the intended bacterial application, and in particular, these conditions have to be kept constant. It must be ensured that small variations in cultivation parameters of bacteria do not change the specified (probiotic) effect on the health of animals and humans. The HPTLC metabolite profiles represented the cultivation conditions of specific bacteria and were found to be a proof of the activity of distinct bacteria. In addition, HPTLC can also be used to optimize and streamline the culture media. The quality control of cultivation or fermentation processes can benefit from such a powerful tool, as a picture is worth a thousand words.