Disentangling de-novo synthesis, recycling and transformation of n-alkyl lipids in soils by combination of position-specific 13C labeling with fragment-specific 13C analysis

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Lipids represent one of the most diverse classes of biomolecules and have various functions: main component of cell membranes, protection layers, storage compounds, etc. High diversity and unique speciation of lipids in plant and microbial taxonomic groups made them to one of the most promising biomarker classes in soils and sediments. They are an important component of soil organic matter (SOM) and contribute to physico-chemical properties like hydrophobicity and C storage. Detailed knowledge on lipid sources exists from studies using lipids as biomarkers, but lipid transformations in soils are nearly unknown. Recycling is assumed to be a highly relevant process in soils as lipids are expensive compounds for de-novo synthesis, but is not proven in-situ, yet. This proposal aims at disentangling microbial transformation of lipid precursors, de-novo synthesis of microbial lipids and recycling of lipids in soil to yield new insights into the network of processes underlying the lipid cycle and to deepen our understanding of SOM transformations. Lipid de-novo synthesis in microbial cells will be investigated by application of 13C glucose, a precursor, which is quickly taken up by microorganisms. Fragment-specific 13C incorporation based on liquid chromatography-mass spectrometry with online fragmentation will enable to separate de-novo synthesis of phospholipid headgroups, backbone and fatty acid alkyl chains. In addition to intracellular de-novo synthesis, lipid recycling from extracellular pools will be investigated. Position-specifically labeled palmitate (13C-1, 13C-2, 13C-16) will be applied to soil and incorporation into microbial phospholipids fatty acids (PLFA) will be analyzed by gas chromatography-mass spectrometry with online fragmentation. Thus, intact incorporation of the added n-alkyl chain into PLFA will reveal recycling of free lipids from soils. The sum of extra- and intracellular transformations of lipids on a medium-term time scale (4 years) will be studied using position-specifically 13C labeled palmitate in an already established field experiment. Recycling and transformations of the n-alkyl chain will be followed into various n-alkyl lipids (n-alkanes, n-alcohols, n-fatty acids, hydroxy fatty acids) of the extractable lipid and hydrolyzable lipid pools. Combining position-specific labeling with fragment-specific 13C incorporation analysis gives us a unique perspective on lipid transformations in soil: Recycling can be unambiguously differentiated from de-novo synthesis in soils in-situ. Distinguishing recycling from stabilization is crucial in understanding the soil C cycle. Such identification of microbial transformations during lipid recycling will strongly improve our interpretation of plant and microbial lipid biomarker fingerprints and deepen our understanding on turnover of one of the major substance classes of SOM.