Construction of soil defined media using quantitative exometabolomic

19 Exometabolomics enables analysis of metabolite utilization of low molecular weight organic 20 substances by soil isolates. Environmentally-based defined media are needed to examine ecologically 21 relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined 22 media using untargeted characterization of water soluble soil metabolites. To broadly characterize soil 23 metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass 24 spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino 25 acids, amino acid derivatives, sugars, sugar alcohols, monoand di-carboxylic acids, osmolytes, 26 nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Water soluble organic 27 carbon was fractionated by molecular weight and measured to determine the fraction of carbon accounted 28 for by the quantified metabolites. This revealed that, much like soil microbial community structures, these 29 soil metabolites have an uneven quantitative distribution, with a single metabolite, trehalose accounting 30 for 9.9 percent of the (< 1 kDa) water extractable organic carbon. This quantitative information was used 31 to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 32 46 (SDM2). To evaluate SDM for supporting the growth of bacteria found at this field site, we examined 33 the growth of 30 phylogenetically diverse soil isolates obtained using standard R2A medium. The simpler 34 SDM1 supported the growth of up to 13 isolates while the more complex SDM2 supported up to 25 35 isolates. One isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series 36 exometabolomics analysis to investigate SDM1 substrate preferences. Interestingly, it was found that this 37 organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose 38 and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results 39 demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally 40 relevant media. We anticipate that this approach can be expanded to other environments to enhance 41 isolation and characterization of diverse microbial communities. 42