Abstract
Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the <1 kDa fraction). This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.
Highlights
Soil organic matter, historically considered to be composed of large polymeric humic substances, is thought to largely consist of microbial products (Schmidt et al, 2011)
Reasoner’s 2A agar medium (R2A) is one of the most widely used for isolations and was developed for the cultivation of bacteria found in potable water (Reasoner and Geldreich, 1985)
Mass spectrometry-based metabolomics methods enable the examination of low molecular weight organic substances (LMWOS) composition, which has revealed a diversity of small molecule metabolites in soils (Warren, 2014; Swenson et al, 2015b)
Summary
Historically considered to be composed of large polymeric humic substances, is thought to largely consist of microbial products (Schmidt et al, 2011). It is not surprising that the water extractable organic carbon (WEOC) (Boyer and Groffman, 1996; Guigue et al, 2014) is associated with high microbial activity and soil respiration (Haney et al, 2012) This reinforces long-standing views that it is desirable for culture media to approximate the conditions, especially in terms of the composition and quantity of metabolites. Mass spectrometry-based metabolomics methods enable the examination of LMWOS composition, which has revealed a diversity of small molecule metabolites in soils (Warren, 2014; Swenson et al, 2015b) Soil metabolomics methods such as these may have utility in informing the development of relevant culture media to support laboratory studies of soil microorganisms
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