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.

The key to reproducible data reduction and data processing in scientific research is the ability to faithfully record every step of the process in a reproducible format that is transparent and easy to communicate. This is not an easy task. Most of the time in experimental research that is not primarily computational in nature, it falls victim to the enormous effort required to design experiments well, run complex analytical procedures rigorously and efficiently, and interpret the results in the proper geologic, geochemical or biological context, with little time left to invest in documenting and constructing a reproducible data reduction workflow. While this is understandable, it introduces a high risk for error, makes it extremely difficult to share and discuss one’s approach or review others’, reproduce the calculations at a later point or even just revisit what was done conceptually. Part of the problem lies inherently with most data processing being difficult to document, part of black box process where the inner workings are inaccessible, or simply too divorced from the narrative of the scientific work it represents. One important obstacle that interferes frequently with attempts to remedy this situation in the stable isotope community is the lack of many basic computational and data access tools that enable the kinds of calculations and data processing isotope geochemists need to do on a day to day basis. Here, we introduce a new suite of software packages that provide efficient and transparent access to raw stable isotope ratio mass spectrometry (IRMS) data formats and enable reproducible data processing straight from raw analytical output through data reduction, quality control, visualization and data reporting that retains the necessary flexibility required for the enormous breadth of analytical goals in the stable isotope community. Presented tools will cover aspects of functionality provided by the isoreader (isoreader.kopflab.org), isoviewer (isoviewer.kopflab.org) and isoprocessor (isoprocessor.kopflab.org) software packages and are 100% open-source and freely available to everyone in the geochemical community and beyond.

In high-pH ($\text{pH}>10$) fluids that have participated in low-temperature ($<150\,^{\circ}\text{C}$) serpentinization, the dominant form of C is often methane (CH$_{4}$), but the origin of this CH$_{4}$ is uncertain. To assess CH$_{4}$ origin during low-temperature serpentinization, we pumped fluids from aquifers within the Samail Ophiolite, Oman. We determined fluid chemical compositions, analyzed taxonomic profiles of fluid-hosted microbial communities, and measured isotopic compositions of hydrocarbon gases. We found that 16S rRNA gene sequences affiliated with methanogens were widespread in the aquifer. We measured clumped isotopologue ($^{13}$CH$_{3}$D and $^{12}$CH$_{2}$D$_{2}$) relative abundances less than equilibrium, consistent with substantial microbial CH$_{4}$ production. Further, we observed an inverse relationship between dissolved inorganic C concentrations and $\delta^{13}\text{C}_{\text{CH}_{4}}$ across fluids bearing microbiological evidence of methanogenic activity, suggesting that the apparent C isotope effect of microbial methanogenesis is modulated by C availability. A second source of CH$_{4}$ is evidenced by the presence of CH$_{4}$-bearing fluid inclusions in the Samail Ophiolite and our measurement of high $\delta^{13}\text{C}$ values of ethane and propane, which are similar to those reported in studies of CH$_{4}$-rich inclusions in rocks from the oceanic lithosphere. In addition, we observed 16S rRNA gene sequences affiliated with aerobic methanotrophs and, in lower abundance, anaerobic methanotrophs, indicating that microbial consumption of CH$_{4}$ in the ophiolite may further enrich CH$_{4}$ in $^{13}$C. We conclude that substantial microbial CH$_{4}$ is produced under varying degrees of C limitation and mixes with abiotic CH$_{4}$ released from fluid inclusions. This study lends insight into the functioning of microbial ecosystems supported by water/rock reactions.