Evaluation of extraction solvents for untargeted metabolomics to decipher the dissolved organic matter of Antarctic cryoconite holes

Cryoconite holes are biological hotspots with a high biogeochemical turnover rate, contributing significantly to the glacial ecosystems overall carbon cycles and net fluxes. Unfortunately, the information about the composition of low molecular weight molecules formed through the metabolic processes of cryoconite-dwelling microbes is scanty. These molecules constitute a substantial portion of the dissolved organic matter (DOM) within cryoconite holes. The present study investigated the composition of DOM in cryoconite holes using reverse-phase liquid chromatography (RP-LC) coupled with high-resolution tandem mass spectrometry. We evaluated various solvent combinations of water, methanol, and acetonitrile to extract chemically diverse polar and non-polar metabolites from the cryoconite holes. Among the single solvents, organic-rich MeOH: Water (70:30 v/v) and in parallel 2-single solvent combinations of MeOH: Water (70:30 v/v) and Acetonitrile: Methanol: Water (40:40:20 v/v) provided increased number and chemical diversity of extracted metabolites. Combining RP with the hydrophilic interaction liquid chromatography (HILIC) technique provided the highest number of unique metabolites. This dual-LC and ionization polarity combination increased the detection of metabolic features by 46.96% and 24.52% in single- and two-solvent combinations compared to RP alone. This study developed a simple untargeted metabolomics workflow that is highly sensitive and robust, detecting and potentially identifying a large number of chemically diverse molecules present in the DOM (extracellular) and microbes (intracellular) from the cryoconite holes environment. This method can better characterize DOMs chemical composition and, after integrating with other omics approaches, can be used to examine the link between metabolic pathways and microbial communities in global cryoconite holes or other similar ecosystems, revealing how these earthy systems and their microbial flora control carbon or nutrient storage or release in response to global climate change. Overall, the study presents a valuable methodology for studying the biogeochemistry of cryoconite holes.