Rationale: Doubly substituted isotope species ("clumped" isotopes) can provide insights into the biogeochemical history of a molecule, including its temperature of formation and/or its (bio)synthetic pathway. Here, we propose a new fluorination method for the measurement of 13 C-13 C species in C 2 molecules using a conventional isotope ratio mass spectrometer. Target molecules include ethane, ethene and ethanol.Methods: 13 C-13 C isotope species in C 2 molecules were measured as C 2 F 6 using a conventional isotope ratio mass spectrometer. Ethane and ethene are directly fluorinated to C 2 F 6 . Ethanol is measured after dehydration to ethene and subsequent fluorination of the latter. The method enables the measurement of the Δ 13 C 13 C values normalized against a reference working standard. Results:The reproducibility of the whole protocol, including chemical modification steps and measurement of C 2 F 6 isotopologues, is better than ±0.14‰ for all the compounds. Ethane from natural gas samples and biologically derived ethanol show a narrow range of Δ 13 C 13 C values, varying from 0.72‰ to 0.90‰. In contrast, synthetic ethanol as well as putative abiotic ethane show Δ 13 C 13 C values significantly different from this range with values of 1.14‰ and 0.25‰, respectively. Conclusions:The method presented here provides alternative means of measuring 13 C-13 C species to that using high-resolution mass spectrometry. Preliminary data from natural and synthetic molecules re-emphasizes the potential of 13 C clumped isotope species as a (bio)marker.

Rationale The 13C‐13C isotopologues of C2 molecules have recently been measured using a fluorination method. The C2 compound is first fluorinated into hexafluoroethane (C2F6), and its 13C‐isotopologues are subsequently measured using a conventional isotope ratio mass spectrometer. Here, we present an approach for standardizing the fluorination method on an absolute reference scale by using isotopically enriched C2F6. Methods We prepared physical mixtures of 13C‐13C‐labeled ethanol and natural ethanol. The enriched ethanol samples were measured using the recently developed fluorination method. Based on the difference between the calculated and measured ∆13C13C values, we quantified the extent to which isotopologues were scrambled during dehydration, fluorination, and ionization in the ion source. Results The measured ∆13C13C value was approximately 20% lower than that expected from the amount of 13C‐13C ethanol. The potential scrambling in the ion source was estimated to be 0.5–2%, which is lower than the observed isotopic reordering. Therefore, isotopic reordering may have occurred during either dehydration or fluorination. Conclusions For typical analysis of natural samples, scrambling in the ion source can only change the ∆13C13C value by less than 0.04‰, which is lower than the current analytical precision (±0.07‰). Therefore, the observed isotopic reordering may have occurred during the fluorination of ethene through the scrambling of isotopologues of ethene but not in the ion source of the mass spectrometer or during the dehydration of ethanol, given the small amount of C1 and C3+ molecules. Thus, we obtained the empirical transfer function ∆13C13CCSC = λ × ∆13C13C with a λ value of 1.25 ± 0.01 for ethanol/ethene and 1.00 for ethane. Using the empirical transfer function, the developed fluorination method can provide actual differences in ∆ values.

Distinguishing biotic compounds from abiotic ones is important in resource geology, biogeochemistry, and the search for life in the universe. Stable isotopes have traditionally been used to discriminate the origins of organic materials, with particular focus on hydrocarbons. However, despite extensive efforts, unequivocal distinction of abiotic hydrocarbons remains challenging. Recent development of clumped-isotope analysis provides more robust information because it is independent of the stable isotopic composition of the starting material. Here, we report data from a 13C-13C clumped-isotope analysis of ethane and demonstrate that the abiotically-synthesized ethane shows distinctively low 13C-13C abundances compared to thermogenic ethane. A collision frequency model predicts the observed low 13C-13C abundances (anti-clumping) in ethane produced from methyl radical recombination. In contrast, thermogenic ethane presumably exhibits near stochastic 13C-13C distribution inherited from the biological precursor, which undergoes C-C bond cleavage/recombination during metabolism. Further, we find an exceptionally high 13C-13C signature in ethane remaining after microbial oxidation. In summary, the approach distinguishes between thermogenic, microbially altered, and abiotic hydrocarbons. The 13C-13C signature can provide an important step forward for discrimination of the origin of organic molecules on Earth and in extra-terrestrial environments.

Understanding hydrocarbon cycling in the subsurface is important in various disciplines including climate science, energy resources and astrobiology. Mud volcanoes provide insights into biogeochemical processes occurring in the subsurface. They are usually associated with natural gas reservoirs consisting mainly of methane and other hydrocarbons as well as CO2. Stable isotopes have been used to decipher the sources and sinks of hydrocarbons in the subsurface, although the interpretation can be ambiguous due to the numerous processes involved. Here we report new data for hydrocarbon isotope analysis, including position-specific isotope composition of propane, for samples from the Tokamachi mud volcano area, Japan. The data suggest that C2+ hydrocarbons are being biodegraded, with indirect production of methane (“secondary methanogenesis”). Data from chemical and isotopic composition are discussed with regard to 16S rRNA analysis, which exhibits the presence of hydrogenotrophic and acetoclastic methoanogens. Overall, the combination of isotopologue analysis with 16S rRNA gene data allows refining of our understanding of hydrocarbon cycling in subsurface environments.

<p>Terrestrial hydrocarbon seeps are widely distributed in oil/gas field. To constrain the sources and post-generation processes occurring in these seeping gases, various geochemical approaches, such as chemical and stable isotope composition (δ<sup>13</sup>C and δD) of hydrocarbons have been extensively used. However, the interpretation can be ambiguous due to the overlap of signatures when using these approaches only<sup>1</sup>. Some recently developed analytical techniques, such as methane clumped isotope analysis (Δ<sup>13</sup>CH<sub>3</sub>D and Δ<sup>12</sup>CH<sub>2</sub>D<sub>2</sub>)<sup>2-4</sup> and propane position-specific isotope analysis (PSIA)<sup>5</sup> may provide new clues to improve our understanding of the origin and fate of hydrocarbons.</p> <p>In this study, we focus on gas samples from different gas seeps and mud volcanos in central Japan collected from 2019 to 2022, where hydrocarbons were considered mainly originating from thermal cracking of organic matter<sup>6</sup>. Gas compositions, bulk stable isotopes of hydrocarbons and associated CO<sub>2</sub>, clumped isotopes of methane, PSIA of propane and other geochemical parameters have been studied. Coupled methane clumped isotope signatures and propane PSIA information provide direct evidence of secondary microbial methane formation associated with biodegradation of non-methane hydrocarbons. The contribution of secondary microbial methane in different seeps/mud volcanos and its temporal changes are also discussed by a mixing model integrating all these isotope information, which provides valuable constraints on methane sources in terrestrial seeps.</p> <p><em>References: [1] Milkov and Etiope, 2018, Org. Geochem.; [2] Stolper et al., 2014, </em><em>Geochim. Cosmochim. Acta.; </em><em>[3] Stolper et al., 2014, Science; [4] Young et al., 2017, </em><em>Geochim. Cosmochim. Acta.; [5]</em><em> Gilbert et al., 2019, Proc. Natl. Acad. Sci. U.S.A.; [6] Etiope et al., 2011, Appl. Geochem.</em></p>