Lipid hydrogen isotope compositions primarily reflect growth water in the model archaeon Sulfolobus acidocaldarius

The stable hydrogen isotope composition (δ ² H) of lipid biomarkers can track environmental processes and remain stable over geologically relevant time scales, enabling studies of past climate, hydrology, and ecology. Most research has focused on lipids from the domain Eukarya (e.g., plant waxes, long-chain alkanes), and the potential of prokaryotic lipid biomarkers from the domain Archaea to offer unique insights into environments not captured by eukaryotic lipids remains unclear. Here, we investigate the H-isotope composition of biphytanes in Sulfolobus acidocaldarius, a model thermoacidophile and obligate heterotroph. We conducted a series of experiments that varied temperature, pH, shaking rate, electron acceptor availability, or electron donor flux. From these experiments, we quantified the lipid/water H-isotope fractionation ( ² ϵ _L/W ) values for core biphytane chains derived from tetraether lipids. The ² ϵ _L/W values are consistently negative (-230 to -180 ‰) and are relatively invariant across all experiments despite the 20-fold change in doubling times and 2-fold change in lipid cyclization. The magnitude and relative invariance of ² ϵ _L/W values are consistent with studies on other heterotrophic archaea and suggests archaeal lipids may be faithful recorders of the δ ² H composition of growth water. Our study highlights the potential of archaeal lipid ² ϵ _L/W as a hydrological proxy, offering new insights into environments where traditional proxies, such as plant-derived lipids, are not available, including extreme environments and extraterrestrial settings.