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
Mineralogy, Petrology and Geochemistry
Final Report Abstract
Lipids are an important component of soil organic matter (SOM) and contribute to physicochemical properties like hydrophobicity and C storage but also represent one of the most promising biomarker classes in soils and sediments. Although lipid sources are already well described, lipid transformations in soils strongly lack understanding. Thus, this project targeted the disentangling of 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 lipid cycling. 14C-/13C-/33P-triple isotope labeling followed by phospholipid molecular moiety specific isotope analysis (14C of headgroup & backbone, 33P of phosphate ester group and 14C & 13C of fatty acids) demonstrated that these phospholipid moieties are rather independent of each other. Whereas headgroups had high turnover and showed rapid de-novo synthesis, the n-alkyl chains of fatty acids were much less isotopically enriched due to a preferred recycling of non-enriched n-alkyl chains. This recycling could also be validated under field conditions, by the microbial use of position-specifically 13C labeled palmitate for the formation of microbial membrane lipids (the phospholipid fatty acids (PLFA)). Equal incorporation of the three 13C enriched positions (C-1, C-2 and C-16) into several odd-numbered PLFA (e.g. 16:1ω7) suggested recycling of the palmitatederived n-alkyl chain. However, incorporation of only C-16 (and not C-1 and C-2) into the saturated 14:0 PLFA demonstrated, that the palmitate-derived n-alkyl chain was oxidized by β-oxidation, which cleaves of a C2-unit at the carboxylic end, and thus formed the 14:0 fatty acid. Thus, microbes recycle intact n-alkyl chains from soil solution, potentially modify them according to their cellular demand, and incorporate them into their phospholipids. Two incubation experiments demonstrated under which environmental conditions recycling and intracellular transformations are of especial importance. Exposure of microbial communities to toxicants, which eliminate a significant proportion of the microbial community, create – once the growth-inhibiting effect of the toxicant is overcome – an environment of low microbial abundance but high C availability (i.e. microbial necromass). The rapid recovery of microbes in toxicant exposed soils could be demonstrated here to be strongly fostered by the recycling of complex metabolites such as lipids, which then do not need to be synthesized costly de-novo. Furthermore, we could demonstrate that rapid adaptations of the membrane fluidity, as required to survive soil temperature changes, can be attributed to a high metabolic capacity of rapid modifications of n-alkyl lipid structures. A deeper insight into microbial lipid formation and transformation processes was gained by establishing a novel gas chromatography-mass spectrometry-based approach for detection of 13C enrichment of fatty acid fragments. For the ethanoate (m/z 74) and propionate (m/z 87) fragments and the molecular ion (M+) of saturated and branched fatty acids, we were able to precisely and accurately determine mass isotope distributions (MID) and 13C fractional enrichments if isotopic enrichment exceeded 0.2 at%. A modified metabolic flux model was programmed, which allowed the quantitative reconstruction of C allocation through glycolysis, pentose phosphate pathway, gluconeogenesis and Entner-Doudoroff pathway based on ethanoate and propionate MID determination of experiments with position-specific glucose labeling. Pure strains and soil samples were analyzed with this new approach and microbial groups of contrasting metabolic flux profiles and carbon use efficiencies could be differentiated in soils. Furthermore, lipid transformations were characterized in field and incubation experiments by application of position-specifically labelled lipid monomers. Palmitate-derived 13C recovery (C-1, C- 2 and C-16) in free fatty acids showed that the microbial palmitate recycling is the major process determining the fate of the palmitate n-alkyl chain. Neutral lipid analysis of the same experiment suggests that reduction of the palmitate carboxyl group to the respective n-alkyl alcohol is an abundant process in soils, whereas the terminal decarboxylation to pentadecane was not detectable. Application of diacids, 13C-labelled at both of their carboxyl groups, suggests chain length being not the only factor determining the persistence of these lipids in soils. Instead microbial preference and transformations have crucial impact on the stabilization of these n-alky lipids, whose esterification to hydrolyzable lipids remains to be quantified. This project developed new methodological approaches and showed first applications for a set of techniques to disentangle biosynthetic processes from recycling and transformations of lipids in soils. Thus, it substantially contributed to the understanding on the turnover of one of the major substance classes of SOM.
Publications
- 2016. Direct incorporation of fatty acids into microbial phospholipids in soils: Position-specific labeling tells the story. Geochimica et Cosmochimica Acta 174, 211-221
Dippold, M.A., Kuzyakov, Y.
(See online at https://doi.org/10.1016/j.gca.2015.10.032) - 2017. Soil microorganisms can overcome respiration inhibition by coupling intra- and extracellular metabolism: C-13 metabolic tracing reveals the mechanisms. ISME Journal 11, 1423-1433
Bore, E.K., Apostel, C., Halicki, S., Kuzyakov, Y., Dippold, M.A.
(See online at https://doi.org/10.1038/ismej.2017.3) - 2018. Food for microorganisms: Position-specific C-13 labeling and C-13-PLFA analysis reveals preferences for sorbed or necromass C. Geoderma 312, 86-94
Apostel, C., Herschbach, J., Bore, E.K., Spielvogel, S., Kuzyakov, Y., Dippold, M.A.
(See online at https://doi.org/10.1016/j.geoderma.2017.09.042) - 2019. Compound-specific C-13 stable isotope probing confirms synthesis of polyhydroxybutyrate by soil bacteria. Rapid Communications in Mass Spectrometry 33, 795-802
Mason-Jones, K., Banfield, C.C., Dippold, M.A.
(See online at https://doi.org/10.1002/rcm.8407) - 2019. Structural and physiological adaptations of soil microorganisms to freezing revealed by position-specific labeling and compound-specific 13 C analysis. Biogeochemistry 143, 207-219
Bore, E.K., Halicki, S., Kuzyakov, Y., Dippold, M.A.
(See online at https://doi.org/10.1007/s10533-019-00558-5)
