Low 13C-13C abundances in abiotic ethane.

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.

Hydrocarbon Cycling in the Tokamachi Mud Volcano (Japan): Insights from Isotopologue and Metataxonomic Analyses.

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.

Depleted carbon isotope compositions observed at Gale crater, Mars.

Obtaining carbon isotopic information for organic carbon from Martian sediments has long been a goal of planetary science, as it has the potential to elucidate the origin of such carbon and aspects of Martian carbon cycling. Carbon isotopic values (δ13CVPDB) of the methane released during pyrolysis of 24 powder samples at Gale crater, Mars, show a high degree of variation (-137 ± 8‰ to +22 ± 10‰) when measured by the tunable laser spectrometer portion of the Sample Analysis at Mars instrument suite during evolved gas analysis. Included in these data are 10 measured δ13C values less than -70‰ found for six different sampling locations, all potentially associated with a possible paleosurface. There are multiple plausible explanations for the anomalously depleted 13C observed in evolved methane, but no single explanation can be accepted without further research. Three possible explanations are the photolysis of biological methane released from the subsurface, photoreduction of atmospheric CO2, and deposition of cosmic dust during passage through a galactic molecular cloud. All three of these scenarios are unconventional, unlike processes common on Earth.

Standardization for 13 C-13 C clumped isotope analysis by the fluorination method.

RATIONALE: The 13 C-13 C isotopologues of C2 molecules have recently been measured using a fluorination method. The C2 compound is first fluorinated into hexafluoroethane (C2 F6 ), and its 13 C-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 C2 F6 . METHODS: We prepared physical mixtures of 13 C-13 C-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 ∆13 C13 C values, we quantified the extent to which isotopologues were scrambled during dehydration, fluorination, and ionization in the ion source. RESULTS: The measured ∆13 C13 C value was approximately 20% lower than that expected from the amount of 13 C-13 C 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 ∆13 C13 C 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 ∆13 C13 CCSC = λ × ∆13 C13 C 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.

A fluorination method for measuring the 13 C-13 C isotopologue of C2 molecules.

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 C2 molecules using a conventional isotope ratio mass spectrometer. Target molecules include ethane, ethene and ethanol. METHODS: 13 C-13 C isotope species in C2 molecules were measured as C2 F6 using a conventional isotope ratio mass spectrometer. Ethane and ethene are directly fluorinated to C2 F6 . Ethanol is measured after dehydration to ethene and subsequent fluorination of the latter. The method enables the measurement of the Δ13 C13 C values normalized against a reference working standard. RESULTS: The reproducibility of the whole protocol, including chemical modification steps and measurement of C2 F6 isotopologues, is better than ±0.14‰ for all the compounds. Ethane from natural gas samples and biologically derived ethanol show a narrow range of Δ13 C13 C values, varying from 0.72‰ to 0.90‰. In contrast, synthetic ethanol as well as putative abiotic ethane show Δ13 C13 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.

Intramolecular isotopic evidence for bacterial oxidation of propane in subsurface natural gas reservoirs.

Microbial anaerobic oxidation of hydrocarbons is a key process potentially involved in a myriad of geological and biochemical environments yet has remained notoriously difficult to identify and quantify in natural environments. We performed position-specific carbon isotope analysis of propane from cracking and incubation experiments. Anaerobic bacterial oxidation of propane leads to a pronounced and previously unidentified 13C enrichment in the central position of propane, which contrasts with the isotope signature associated with the thermogenic process. This distinctive signature allows the detection and quantification of anaerobic oxidation of hydrocarbons in diverse natural gas reservoirs and suggests that this process may be more widespread than previously thought. Position-specific isotope analysis can elucidate the fate of natural gas hydrocarbons and provide insight into a major but previously cryptic process controlling the biogeochemical cycling of globally significant greenhouse gases.

Geoelectrochemical CO production: Implications for the autotrophic origin of life.

Wächtershäuser's proposal of the autotrophic origin of life theory and subsequent laboratory demonstrations of relevant organic reactions have opened a new gate for the exploration of the origin of life. However, this scenario remains controversial because, at present, it requires a high pressure of CO as a source of carbon and reducing energy, although CO must have been a trace C species on the Hadean Earth. We show that, simulating a geoelectrochemical environment in deep-sea hydrothermal fields, CO production with up to ~40% Faraday efficiency was attainable on CdS in CO2-saturated NaCl solution at ≤-1 V (versus the standard hydrogen electrode). The threshold potential is readily generated in the H2-rich, high-temperature, and alkaline hydrothermal vents that were probably widespread on the early komatiitic and basaltic ocean crust. Thus, Wächtershäuser's scenario starting from CO2 was likely to be realized in the Hadean ocean hydrothermal systems.

Expanded uncertainty associated with determination of isotope enrichment factors: Comparison of two point calculation and Rayleigh-plot.

The enrichment factor (ε) is a common way to express Isotope Effects (IEs) associated with a phenomenon. Many studies determine ε using a Rayleigh-plot, which needs multiple data points. More recent articles describe an alternative method using the Rayleigh equation that allows the determination of ε using only one experimental point, but this method is often subject to controversy. However, a calculation method using two points (one experimental point and one at t0) should lead to the same results because the calculation is derived from the Rayleigh equation. But, it is frequently asked "what is the valid domain of use of this two point calculation?" The primary aim of the present work is a systematic comparison of results obtained with these two methodologies and the determination of the conditions required for the valid calculation of ε. In order to evaluate the efficiency of the two approaches, the expanded uncertainty (U) associated with determining ε has been calculated using experimental data from three published articles. The second objective of the present work is to describe how to determine the expanded uncertainty (U) associated with determining ε. Comparative methodologies using both Rayleigh-plot and two point calculation are detailed and it is clearly demonstrated that calculation of ε using a single data point can give the same result as a Rayleigh-plot provided one strict condition is respected: that the experimental value is measured at a small fraction of unreacted substrate (f < 30%). This study will help stable isotope users to present their results in a more rigorous expression: ε ± U and therefore to define better the significance of an experimental results prior interpretation. Capsule: Enrichment factor can be determined through two different methods and the calculation of associated expanded uncertainty allows checking its significance.

The Abiotic Chemistry of Thiolated Acetate Derivatives and the Origin of Life.

Thioesters and thioacetic acid (TAA) have been invoked as key reagents for the origin of life as activated forms of acetate analogous to acetyl-CoA. These species could have served as high-energy group-transfer reagents and allowed carbon insertions to form higher molecular weight compounds such as pyruvate. The apparent antiquity of the Wood-Ljungdahl CO2 fixation pathway and its presence in organisms which inhabit hydrothermal (HT) environments has also led to suggestions that there may be a connection between the abiotic chemistry of compounds similar to TAA and the origins of metabolism. These compounds' apparent chemical simplicity has made their prebiotic availability assumed, however, although the kinetic behavior and thermochemical properties of TAA and analogous esters have been preliminarily explored in other contexts, the geochemical relevance of these compounds merits further evaluation. Therefore, the chemical behavior of the simplest thiolated acetic acid derivatives, TAA and methylthioacetate (MTA) were explored here. Using laboratory measurements, literature data, and thermochemical models, we examine the plausibility of the accumulation of these compounds in various geological settings. Due to the high free energy change of their hydrolysis and corresponding low equilibrium constants, it is unlikely that these species could have accumulated abiotically to any significant extant.

Evaluation of on-line pyrolysis coupled to isotope ratio mass spectrometry for the determination of position-specific (13)C isotope composition of short chain n-alkanes (C6-C12).

We measured (13)C intramolecular isotopic composition of commercially available short-chain hydrocarbons (n-C6-n-C12) using (13)C-NMR. Results show that the main variation is between the terminal and the sub-terminal C-atom positions. Site-preference (difference in δ(13)C values between terminal and sub-terminal C-atom positions) among all the samples varies between -12.2‰ and +8.4‰. Comparison of these results with those obtained using on-line pyrolysis coupled with GC-C-IRMS show that the thermal cracking of hydrocarbons occurs with a good isotopic fidelity between terminal and sub-terminal C-atom positions of the starting material and the related pyrolysis products (methane and ethylene). On-line pyrolysis coupled with GC-C-IRMS can thus be used for tracing hydrocarbons biogeochemical processes.

Measurement of natural carbon isotopic composition of acetone in human urine.

The natural carbon isotopic composition of acetone in urine was measured in healthy subjects using gas chromatography-combustion-isotope ratio mass spectrometry combined with headspace solid-phase microextraction (HS-SPME-GC-C-IRMS). Before applying the technique to a urine sample, we optimized the measurement conditions of HS-SPME-GC-C-IRMS using aqueous solutions of commercial acetone reagents. The optimization enabled us to determine the carbon isotopic compositions within ±0.2 ‰ of precision and ±0.3‰ of error using 0.05 or 0.2 mL of aqueous solutions with acetone concentrations of 0.3-121 mg/L. For several days, we monitored the carbon isotopic compositions and concentrations of acetone in urine from three subjects who lived a daily life with no restrictions. We also monitored one subject for 3 days including a fasting period of 24 h. These results suggest that changes in the availability of glucose in the liver are reflected in changes in the carbon isotopic compositions of urine acetone. Results demonstrate that carbon isotopic measurement of metabolites in human biological samples at natural abundance levels has great potential as a tool for detecting metabolic changes caused by changes in physiological states and disease.

Analytical method for simultaneous determination of bulk and intramolecular (13) C-isotope compositions of acetic acid.

RATIONALE: Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography/pyrolysis-gas chromatography/combustion-isotope ratio mass spectrometry (GC/Py-GC/C-IRMS) was developed for the simultaneous determination of the intramolecular and molecular carbon-isotopic composition (δ(13) C value) of acetic acid. METHODS: The δ(13) C values of carboxyl and methyl carbon were standardized using calibration curves constructed from the regression between the measured δ(13) C values and the δ(13) C values of working standards determined in a previous study. We applied this developed HS-SPME-GC/Py-GC/C-IRMS technique to commercial vinegars. RESULTS: In one injection analysis, the bulk and intramolecular δ(13) C values of pure acetic acid standards can be obtained. The repeatability (1σ) of the bulk δ(13) C values is within ±0.4‰, and that of the δ(13) Ccarboxyl and δ(13) Cmethyl values is within ±0.6‰. The intramolecular δ(13) C values of acetic acid in vinegars exhibit a similar pattern. The average Δδ value (δ(13) CCOOH - δ(13) CCH3 ) is 4.3 ± 2.0‰. CONCLUSIONS: The approach presented herein for the molecular and intramolecular δ(13) C determination of acetic acid avoids switching between configuration systems and thereby reduces systematic errors. It is expected to be useful for examining isotope fractionation associated with processes related to organic acid (bio)transformations.

Position-Specific Isotope Analysis of Xanthines: A (13)C Nuclear Magnetic Resonance Method to Determine the (13)C Intramolecular Composition at Natural Abundance.

The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine.

Determination of carbon isotopic measurement conditions for ceramide in skin using gas chromatography-combustion-isotope ratio mass spectrometry.

The ceramide (Cer) content of skin and glucosylceramide (GlcCer) intake affect skin moisture conditions, but their mutual relation in skin remains unclear. For clarification of that mutual relation, carbon stable isotopes ((12)C and (13)C) are useful as a tracer. However, carbon isotopic measurement has not been applied to the study of clarifying their skin moisturizing effects. Therefore, we used gas chromatography / combustion / isotope ratio mass spectrometry (GC-C-IRMS) to ascertain the appropriate conditions for carbon isotopic measurements using synthesized Cer (SCer) in substitution for very low concentrations of Cer in skin. SCer was derivatized to trimethylsilylated SCer (TMS-SCer) quantitatively using N-trimethylsilylimidazole (TMSI) depending on the amount of SCer. The derivatization rates were 75-85%. Excess TMSI was removed using three cycles of hexane-water distribution. Under these conditions, carbon isotopic measurements of TMS-SCer conducted using GC-C-IRMS showed high repeatability and good inter-day variation (S.D. < 0.3‰). The carbon stable isotope ratio value (δ(13)C) of SCer calculated using a mass balance equation was compared with δ(13)C of underivatized SCer, which was regarded as the actual δ(13)C of SCer obtained using sealed tube combustion method. The difference between the calculated δ(13)C of SCer and δ(13)C of the underivatized SCer depended on the TMSI reagent supplier and on the number of hydroxyl groups to be derivatized in SCer. For accurate δ(13)C of Cer in skin using GC-C-IRMS, the measured δ(13)C of a target TMS-Cer must be calculated using a correction factor representing the difference in δ(13)C of underivatized standard SCer from that of TMS-standard SCer having a structure resembling that of the target Cer in skin. In addition, we show that the same lot of TMSI reagent from a specific supplier must be used throughout the experiments.

Conditions to obtain precise and true measurements of the intramolecular 13C distribution in organic molecules by isotopic 13C nuclear magnetic resonance spectrometry.

Intramolecular (13)C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic (13)C NMR spectrometry provides a general tool for measuring the position-specific (13)C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal (13)C distribution, and (ii) an approach to determining the "absolute" position-specific (13)C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the (13)C frequency range of the studied molecule, i.e. the chemical shift range. The "absolute value" and, therefore, the trueness of the (13)C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH3 by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py-irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py-irm-MS (thus, the "true" value) while the other showed a bias, which was dependent to the range covered by the resonance frequencies of the molecule. Therefore, the former can be used directly for studying isotope affiliations, while the latter can only be used directly for comparative data, for example in authenticity studies, but can also be used to obtain the true values by applying appropriate correction factors. The present study assesses several key protocol steps required to enable the determination of position-specific (13)C content by isotopic (13)C NMR, irrespective of the NMR spectrometer: parameters to be adjusted, performance test using [1,2-(13)C2]acetic acid, generation of correction factors.

Evaluation of commercially available reagents as a reference material for intramolecular carbon isotopic measurements of acetic acid.

RATIONALE: Recent advances in analytical techniques for the intramolecular carbon isotopic ratio measurement of some organic compounds have provided important information on carbon cycles in biochemistry, organic geochemistry and food chemistry. These advances have made it necessary to prepare intramolecular isotopic reference materials (RMs) to use for inter-laboratory calibration and/or inter-calibration among different analytical methods. METHODS: We evaluated the feasibility of preparing RMs using commercially available reagents for intramolecular carbon isotopic ratio measurement of acetic acid. The intramolecular carbon isotopic distribution of nine acetic acid and four sodium acetate reagents was determined with high precision using off-line pyrolysis combined with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We also evaluated the potential alteration in the isotopic signature of acetic acid reagents by evaporation. RESULTS: The intramolecular carbon isotopic distributions for the acetic acid and sodium acetate reagents were determined with a precision of better than 0.45‰. We found that the isotopic values of these reagents spanned the carbon isotopic range of acetic acid in biological and environmental samples. We also found that the isotope fractionation associated with the evaporation of acetic acid occurs solely on the methyl position, the carboxyl position being unaffected. CONCLUSIONS: These commercially available reagents will be used as RMs in the future for inter-laboratory calibration and/or inter-calibration with another intramolecular isotopic measurement technique, namely quantitative (13) C NMR. In cases where acetic acid is being used as a RM, its storage must be carefully controlled to prevent evaporation.

Site-specific 13C content by quantitative isotopic 13C nuclear magnetic resonance spectrometry: a pilot inter-laboratory study.

Isotopic (13)C NMR spectrometry, which is able to measure intra-molecular (13)C composition, is of emerging demand because of the new information provided by the (13)C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic (13)C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular (13)C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic (13)C NMR was then assessed on vanillin from three different origins associated with specific δ (13)Ci profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ (13)Ci in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.

Accurate method for the determination of intramolecular 13C isotope composition of ethanol from aqueous solutions.

A new method, combining headspace solid phase microextraction (HS-SPME) with an online pyrolysis system coupled with isotope ratio mass spectrometry (IRMS), is developed for the determination of the intramolecular (13)C isotope composition of ethanol in aqueous solutions. The δ(13)C values of the pyrolytic fragments (CO, CH4, C2H4) are shown to be highly reproducible (sd <0.4‰). Furthermore, using 14 ethanol samples of known intramolecular isotope distribution, the CO and CH4 fragments are shown to arise solely from the methylene (CH2OH) and methyl (CH3) carbon atom positions of the original ethanol, respectively. Although the different steps (extraction and pyrolysis) fractionate between (12)C and (13)C, the isotopic fractionation is reproducible (sd <0.4‰), allowing correcting factors to be applied in order to back-calculate the original δ(13)CCH2OH and δ(13)CCH3 values of ethanol. The method thus allows the determination of the isotope composition of ethanol at the intramolecular and molecular levels, within a single run and a short experimental time (30 min), and with a very easy sample preparation. The method is then applied to alcoholic beverages to show its potential for authentication purposes.