Abstract
Soils harbor complex biological processes intertwined with metabolic inputs from microbes and plants. Measuring the soil metabolome can reveal active metabolic pathways, providing insight into the presence of specific organisms and ecological interactions. A subset of the metabolome is volatile; however, current soil studies rarely consider volatile organic compounds (VOCs), contributing to biases in sample processing and metabolomic analytical techniques. Therefore, we hypothesize that overall, the volatility of detected compounds measured using current metabolomic analytical techniques will be lower than undetected compounds, a reflection of missed VOCs. To illustrate this, we examined a peatland metabolomic dataset collected using three common metabolomic analytical techniques: nuclear magnetic resonance (NMR), gas chromatography-mass spectroscopy (GC-MS), and fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). We mapped the compounds to three metabolic pathways (monoterpenoid biosynthesis, diterpenoid biosynthesis, and polycyclic aromatic hydrocarbon degradation), chosen for their activity in peatland ecosystems and involvement of VOCs. We estimated the volatility of the compounds by calculating relative volatility indices (RVIs), and as hypothesized, the average RVI of undetected compounds within each of our focal pathways was higher than detected compounds (p< 0.001). Moreover, higher RVI compounds were absent even in sub-pathways where lower RVI compounds were observed. Our findings suggest that typical soil metabolomic analytical techniques may overlook VOCs and leave missing links in metabolic pathways. To more completely represent the volatile fraction of the soil metabolome, we suggest that environmental scientists take into consideration these biases when designing and interpreting their data and/or add direct online measurement methods that capture the integral role of VOCs in soil systems.
Highlights
As a complex and heterogeneous ecosystem, soil harbors a myriad of biological processes that are challenging to uncover
We show that compounds with high volatility are disproportionately undetected in a peatland metabolomic dataset derived from three techniques (GC-MS, Fourier-transform ion cyclotron resonance MS [FTICR-MS] by direct injection, and nuclear magnetic resonance [NMR])
We selected three Volatile organic compounds (VOCs)-containing pathways that we expect to be present in peatland ecosystems: (1) monoterpenoid biosynthesis (Mono Bio), describing the formation of monoterpenes which are highly volatile; (2) diterpenoid biosynthesis (Di Bio), describing the formation of diterpenes which include many nonvolatile compounds with a few exceptions; and (3) polycyclic aromatic hydrocarbon degradation (PAH Deg), describing the breakdown of hydrocarbons and including many semi-volatile compounds
Summary
As a complex and heterogeneous ecosystem, soil harbors a myriad of biological processes that are challenging to uncover. Inherent biases, including target size of compounds and ionization mode (positive vs negative), exist for each of the most widely used metabolomic analysis techniques (Table 1) Some of these challenges to measuring the soil volatilome may be addressed by adapting online measurement methods from atmospheric chemistry that directly measure VOCs in the gas phase such as proton-transfer-reaction time of flight MS (PTR-TOF-MS). While the rhizosphere zone influenced by root exudates may be restricted to millimeter-scales for non-volatile compounds, VOCs may diffuse centimeters or farther from roots, thereby extending the reach of the effective rhizosphere (de la Porte et al, 2020) These examples emphasize some of the unique roles of VOCs in the soil and signify that capturing VOCs within the complete soil metabolome is important for resolving belowground processes and aboveground interactions. To predict the volatility of metabolites along metabolic pathways, we adapted tools used to estimate VOC partitioning
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