A defined medium based on R2A for cultivation and exometabolite profiling of soil bacteria

Summary Exometabolomics is an approach to assess how microorganisms alter their environments through the depletion and secretion of chemical compounds. Comparisons of inoculated with uninoculated media can be used to provide direct biochemical observations on depleted and secreted metabolites which can be used to predict resource competition, cross-feeding and secondary metabolite production in microbial isolates and communities. This approach is most powerful when used with defined media that enable tracking of all depleted metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through LC-MS/MS. Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse bacteria but is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 53 phylogenetically diverse soil bacterial isolates and all of its metabolites were trackable through LC–MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for cultivating and characterizing diverse microbial isolates and communities. Originality-Significance Statement We build a defined medium based on the metabolite composition of R2A medium and soil, elemental stoichiometry requirements, and knowledge of metabolite usage by different bacteria. The newly formulated defined medium was evaluated on its ability to support the growth of soil isolates and its application for metabolite utilization profiling. We found that of 53 phylogenetically diverse soil bacterial isolates grew on the defined medium and all of its metabolites were trackable through LC–MS/MS analysis. This demonstrates the viability and utility of the constructed defined medium for cultivating and characterizing diverse microbial isolates and communities.