Metabolic imprints in the hydrogen isotopes of Archaeoglobus fulgidus tetraether lipids

The stable hydrogen isotope composition of archaeal lipids is emerging as a potential paleoenvironmental proxy, adding to the well-established application of plant leaf wax-derived n-alkanes in paleohydrological reconstruction. A handful of studies reported relatively invariant and depleted hydrogen isotope compositions for archaeal lipids despite the range of different organisms and growth conditions explored. However, how modes of metabolism and physiological state (growth phase) affect the hydrogen isotope signatures of archaeal lipids remains poorly understood, limiting our ability to interpret archaeal lipid biomarker records from the environment. Here we conducted water isotope label experiments with a metabolically flexible and well-studied model archaeon Archaeoglobus fulgidus and quantified the hydrogen isotope fractionation between lipids and water in response to different carbon substrates and electron donor–acceptor pairs at different growth phases. The ²H/¹H fractionation between lipids and water (ε_L/W) was overall negative. Both carbon metabolism and growth phase affected the magnitude of isotope fractionation in A. fulgidus; however, the changes in ε_L/W values were relatively subtle where they ranged from –283 to –229 ‰ across all tested conditions, overlapping with the ranges observed for other archaea in previous studies. Isotope flux-balance model results suggest that ≥ 80 % and ≥ 50 % of lipid-bound H in A. fulgidus cultures directly reflect water isotope compositions (i.e., not via organic substrate or H₂) during autotrophy and heterotrophy, respectively. The model results also suggest two main mechanisms of consistent ²H depletion observed in A. fulgidus tetraethers as well as other archaeal lipids reported in previous studies: 1) isotopic re-equilibration via upstream isomerization reactions involving C₅ units and 2) downstream double bond reduction catalyzed by a flavoenzyme geranylgeranyl reductase. These results are consistent with previous isotope flux-balance model results for a different archaeon. Finally, we synthesized available data to compare ε_L/W patterns across all three domains of life: Eukarya, Archaea and Bacteria. Because they vary fundamentally in lipid biosynthesis pathways, we present comparative discussions in pairs, focusing on the shared biochemical mechanisms among isoprenoid lipids and potential signals of metabolic adaptations across prokaryotic lipids. Emerging patterns between diverse archaeal and eukaryotic isoprenoid lipids are consistent with the two proposed mechanisms for ²H depletion identified (isomerization and final saturation). The patterns between archaeal isoprenoids and bacterial fatty acids suggest that the general state of energy limitation may also contribute to large, negative values of ε_L/W observed in prokaryotic lipids. Altogether, these findings lend further support for the potential of archaeal lipid ε_L/W as a paleohydrological proxy and provide a broader insight into the ²H/¹H fractionation mechanisms potentially shared among prokaryotic and eukaryotic lipid biomarkers.