How to reveal the bacterial imprint in natural dissolved organic matter: Optimizing ultrahigh resolution mass spectrometry analysis

Marine dissolved organic matter (DOM) comprises the largest standing stock of reduced carbon in the ocean, yet it is a complex mixture of thousands of molecules with low concentrations. Bacteria are said to play a key role in sustaining the chemodiversity of DOM in the ocean. Bacterial exometabolomes are highly variable in molecular composition, depending on strain, substrate and growth conditions. So far, we lack direct evidence of the contribution to marine DOM from specific bacteria. Here, we tested whether certain exometabolites or characteristic exometabolite signatures can be directly detected in natural DOM via Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS). We used routine and adjusted instrument settings for the molecular analysis of mixtures of DOM extracted from the deep ocean and from culture media of selected model strains of the Roseobacter group that had been grown under defined conditions in the lab. Under standard instrument settings and mixing ratios closest to the natural environment (where the exometabolome was mixed 1:100 with marine DOM), the exometabolome signature was hard to distinguish from marine DOM. However, the total number of detected exometabolites and relative abundance of the exometabolome within the marine DOM background increased with the number of accumulated scans. Moreover, windowing a narrow mass range that encompassed a representative fraction of exometabolites was a particularly promising technique for detecting the bacterial signature. We further tested whether the detection of individual exometabolites is related to their molecular characteristics. Overall, distinguishing the exometabolome from marine DOM was limited by dilution, resolution and narrow peak distribution relative to the large marine DOM background. However, when considering certain parameters, non-targeted FT-ICR-MS analysis of natural DOM can discern individual bacterial exometabolites from the background of natural DOM.