Unraveling the structure and function of soil food webs using an untargeted lipidomics approach

Belowground (soil) communities are highly diverse and encompass higher plants, bacteria, fungi, protists, invertebrates and vertebrates. The feeding relationships are as diverse, ranging from symbiotic associations (e.g. mycorrhizae), saprotrophs, grazers, shredders, predators and parasites. These material flows underly the biogeochemical functions of soils, driving organic matter decomposition, soil carbon sequestration, nutrient recycling and greenhouse gas emissions. Despite the importance of understanding the structure and dynamics of such complex soil food webs we are still lacking quantitative and detailed approaches to characterize them. Recently lipidomics analysis of intact polar lipids of soil communities has emerged indicating its potential to allow disentangling the food web structure beyond just abundances of bacteria and fungi based on phospholipid fatty acids or amplicon sequencing data, but extending this analysis across the whole soil food web including its base, higher plants, and including higher consumer levels with diverse protists and invertebrates Our study introduces and develops an untargeted lipidomics platform, employing reversed-phase liquid chromatography and electrospray ionization tandem mass spectrometry (UPLC ESI Orbitrap MS), to examine the intact polar lipidomes of soil biota. With advanced high resolution mass spectrometry and a newly adopted bioinformatics toolbox, we analyze lipidomes from complex soil communities and from pure cultures and single species, including plants, archaea, bacteria, fungi, protists (amoebozoa, ciliophora, cercozoa, etc.), collembola, mites, nematodes, and other soil fauna, as well as their diets. Our workflow facilitates the rapid identification and quantification of thousands of unique intact polar lipid molecules, representing a variety of biological classes, which we currently analyze for their biomarker potential, for being indicative for the presence and activity of specific groups of soil organisms. Utilizing this method of biomarker analysis, finally in combination with isotopic tracing into the fatty acyl residues (containing carbon, hydrogen and oxygen) and the lipid head groups (containing additionally nitrogen, sulfur and phosphorus), is expected to provide valuable quantitative insights into the structure of soil food webs and their activity and matter transfer, by following the incorporation and transfer of isotopically labeled matter and how this responds to climate and land use change. Thereby we foresee to improve our understanding of the contributions made by soil organisms to the stability and function of soil ecosystems, thus providing a foundation for ongoing ecological and environmental research.