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

脂类是生物分子中种类最多的一类，具有多种功能：细胞膜的主要成分、保护层、储藏化合物等。在植物和微生物分类类群中，脂类的高度多样性和独特的物种组成使其成为土壤和沉积物中最有前途的生物标志物之一。它们是土壤有机质(SOM)的重要组成部分，对疏水性和碳贮量等物理化学性质有重要作用。使用脂类作为生物标志物的研究提供了关于脂类来源的详细知识，但土壤中的脂类转化几乎是未知的。回收在土壤中被认为是一个高度相关的过程，因为脂类是从头合成的昂贵化合物，但尚未在现场得到证实。这一建议旨在解开脂前体的微生物转化、微生物脂的从头合成和土壤中脂的循环，以期对脂类循环的过程网络产生新的见解，并加深我们对SOM转化的理解。微生物细胞中的脂质从头合成将通过应用13C葡萄糖进行研究，13C葡萄糖是一种被微生物迅速吸收的前体。基于在线裂解的液-质联用片段专一性~(13)C掺入法，可以分离磷脂头基、主链和脂肪酸烷基链的从头合成。除了细胞内从头合成外，还将研究从细胞外池中回收脂类。定位标记棕榈酸酯(13C-1，13C-2，13C-16)将应用于土壤，并将结合微生物磷脂脂肪酸(PLFA)的在线裂解气相色谱-质谱仪(GC-MS)分析。因此，将添加的正构烷基链完整地结合到PLFA中将揭示土壤中游离脂的循环。在已经建立的现场实验中，将使用位置特定的13C标记棕榈酸酯来研究中期时间尺度(4年)上脂质的细胞外和细胞内转化的总和。正构烷基链的循环和转化将被转化为可提取的脂类和可水解性脂类的各种正构烷基脂(正构烷烃、正构醇、正脂肪酸、羟基脂肪酸)。结合位置特异性标记和片段特异性13C掺入分析，我们对土壤中的脂肪转化有了一个独特的视角：土壤中的再循环可以明确地与原位从头合成区分开来。区分循环和稳定是理解土壤碳循环的关键。这种对脂肪循环过程中微生物转化的识别将极大地改进我们对植物和微生物脂肪生物标志物指纹的解释，并加深我们对SOM主要物质类别之一周转的理解。

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.