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

The stable hydrogen isotope composition (δ²H) 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 (²ε_L/W) values for core biphytane chains derived from tetraether lipids. The ²ε_L/W values are consistently negative (-230‰ to -180‰) and are relatively invariant across all experiments despite a 20-fold change in doubling times and a twofold change in lipid cyclization. The magnitude and relative invariance of ²ε_L/W values are consistent with studies on other heterotrophic archaea and suggest archaeal lipids may be faithful recorders of the δ²H composition of growth water. Our study highlights the potential of archaeal lipid δ²H values 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.IMPORTANCEReconstructing past climates is crucial for understanding Earth's environmental history and its responses to changing conditions. This study examines Sulfolobus acidocaldarius, a thermoacidophilic archaeon that thrives in extreme environments like hot springs. These microorganisms incorporate hydrogen water in the growth environment into membrane lipids, creating hydrogen isotope signatures that can reflect hydroclimate conditions. Our findings show that these hydrogen isotope ratios remain consistent even under varying temperatures, pH, oxygen levels, and electron donor fluxes, indicating a stable fractionation between lipids and water. This invariance suggests that S. acidocaldarius lipids could serve as a robust proxy for reconstructing ancient water H-isotope values, especially in extreme environments where traditional proxies, such as plant waxes, are absent. This research has broader implications for planetary-scale reconstructions, including potential applications in studying past climates on other planets, such as Mars, where similar microorganisms may have existed in hydrothermal conditions.