The stable hydrogen isotope composition of persistent biomolecules is; used as a paleoenvironmental proxy. While much previous work has focused; on plant leaf wax-derived n-alkanes, the potential of bacterial and; archaeal lipid biomarkers as carriers of H isotope signatures remains; underexplored. Here we investigated H isotope distributions in the; membrane lipids of the ammonia-oxidizing chemoautotroph Nitrosopumilus; maritimus strain SCM1. Hydrogen isotope ratios were measured on the; biphytane chains of tetraether membrane lipids extracted from; steady-state continuous cultures cultivated at slow, medium, and fast; growth rates. In contrast to recent work on bacterial fatty acids, where; the direction and magnitude of isotopic fractionation varies widely (ca.; 600 ‰ range) in response to the choice of substrate and pathways of; energy metabolism, archaeal biphytane data in the present work are; relatively invariant. The weighted average 2H/1H fractionation values; relative to growth water (2εL/W) only ranged from 272 to 260 ‰, despite; a three-fold difference in doubling times (30.8 hr to 92.5 hr), yielding; an average growth-rate effect of 0.2 ‰ hr-1. These 2εL/W values are more; depleted than all heterotrophic archaeal and bacterial lipid H isotope; measurements in the literature, and on par with those from other; autotrophic archaea, as well as isoproenoid-based lipids in; photoautotrophic algae. N. maritimus values of 2εL/W also varied; systematically with the number of internal rings (cyclopentyl +; cyclohexyl), increasing for each additional ring by 6.4 ± 2.7 ‰. Using; an isotope flux-balance model in tandem with a comprehensive analysis of; the sources of H in archaeal lipid biosynthesis, we use this observation; to estimate the kinetic isotope effects (KIEs) of H incorporation from; water; from reducing cofactors such as ferredoxin, and for the; transhydrogenation reaction(s) that convert the electron-donor derived; NADH into NADPH for anabolic reactions. Consistent with prior studies on; bacteria, our results indicate the KIEs of reducing cofactors and; transhydrogenation processes in archaea are highly fractionating, while; those involving exchange of water protons are less so. When combined; with the observation of minimal growth-rate sensitivity, our results; suggest biphytanes of autotrophic 3HP/4HB Thaumarchaeota may be offset; from source waters by a nearly constant 2εL/W value. Together with the; ring effect, this implies that all biphytanes originating from a common; source should have a predictable ordering of their isotope ratios with; respect to biphytane ring number, allowing precise reconstruction of the; original δ2H value of the growth water. Collectively, these patterns; indicate archaeal biphytanes have potential as paleo-hydrological; proxies, either as a complement or an alternative to leaf wax n-alkanes.
