Search

Microbial coal-bed methane is an important economic resource and source of a potent greenhouse gas, but controls on its formation are poorly understood. To test whether the microbial degradability of coal limits microbial methane, we monitored methoxyl group demethylation—a reaction that feeds methanogenesis—in a global sample suite ranging in maturity from wood to bituminous coal. Carbon isotopic compositions of residual methoxyl groups were inconsistent with a thermal reaction, instead implying a substrate-limited biologic process. This suggests that deep biosphere communities participated in transforming plant matter to coal on geologic time scales and that methoxyl abundance influences coal-bed methane yield. Carbon isotopic enrichments resulting from microbial methylotrophy also explain an enigmatic offset in the carbon-13 content of microbial methane from coals and conventional hydrocarbon deposits.

The modern Pacific Ocean hosts the largest oxygen-deficient zones (ODZs), where oxygen concentrations are so low that nitrate is used to respire organic matter. The history of the ODZs may offer key insights into ocean deoxygenation under future global warming. In a 12-My record from the southeastern Pacific, we observe a >10‰ increase in foraminifera-bound nitrogen isotopes (15N/14N) since the late Miocene (8 to 9 Mya), indicating large ODZs expansion. Coinciding with this change, we find a major increase in the nutrient content of the ocean, reconstructed from phosphorus and iron measurements of hydrothermal sediments at the same site. Whereas global warming studies cast seawater oxygen concentrations as mainly dependent on climate and ocean circulation, our findings indicate that modern ODZs are underpinned by historically high concentrations of seawater phosphate., Proceedings of the National Academy of Sciences of the United States of America, 119 (45), ISSN:0027-8424, ISSN:1091-6490

One of the outstanding current problems in both geobiology and environmental microbiology is the quantitative analysis of in situ microbial metabolic activities. Techniques capable of such analysis would have wide application, from quantifying natural rates of biogeochemical cycling to identifying the metabolic activity of uncultured organisms. We describe here a method that represents one step towards that goal, namely the high-precision measurement of ^(13)C in specific populations of microbial cells that are purified by fluorescence-activated cell sorting (FACS). Sorted cells are concentrated on a Teflon membrane filter, and their ^(13)C content is measured by coupling an isotope ratio mass spectrometer (IRMS) with a home-built spooling wire microcombustion (SWiM) apparatus. The combined instrumentation provides measurements of δ^(13)C in whole cells with precision better than 0.2‰ for samples containing as little as 25 ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 10^4 eukaryotic or 10^7 bacterial cells per sample. Coupled with ^(13)C-labelled substrate additions, this approach has the potential to directly quantify uptake of metabolites in specific populations of sorted cells. The high precision afforded by SWiM-IRMS also permits useful studies of natural abundance variations in ^(13)C. The approach is equally applicable to specific populations of cells sorted from multicellular organisms.

The rapid turnover of dimethylsulfoniopropionate (DMSP), likely the most relevant dissolved organic sulfur compound in the surface ocean, makes it pivotal to understand the cycling of organic sulfur. Dimethylsulfoniopropionate is mainly synthesized by phytoplankton, and it can be utilized as carbon and sulfur sources by marine bacteria or cleaved by bacteria or algae to produce the volatile compound dimethylsulfide (DMS), involved in the formation of sulfate aerosols. The fluxes between the consumption (i.e., demethylation) and cleavage pathways are thought to depend on community interactions and their sulfur demand. However, a quantitative assessment of the sulfur partitioning between each of these pathways is still missing. Here, we report for the first time the sulfur isotope fractionations by enzymes involved in DMSP degradation with different catalytic mechanisms, expressed heterologously in Escherichia coli. We show that the residual DMSP from the demethylation pathway is 2.7‰ enriched in δ ³⁴S relative to the initial DMSP, and that the fractionation factor (³⁴ε) of the cleavage pathways varies between −1 and −9‰. The incorporation of these fractionation factors into mass balance calculations constrains the biological fates of DMSP in seawater, supports the notion that demethylation dominates over cleavage in marine environments, and could be used as a proxy for the dominant pathways of degradation of DMSP by marine microbial communities.

Rationale: Position-specific ¹³C/¹²C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids. Methods: Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis(trifluoroacetyl)methyl ester) was isolated using gas chromatography, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The ¹³C/¹²C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ¹³C values relative to a working standard. Results: This methodology constrains position-specific δ¹³C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ¹³C values of fragment ions of serine were characterized with ≤1‰ precisions, leading to propagated standard errors of 0.7–5‰ for each carbon position. Position-specific δ¹³C values differed by up to ca 28 ± 5‰ between serine molecules hydrolyzed from plants grown under contrasting pCO₂, selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline. Conclusions: This study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.

The Thermo Scientific™ QExactive Orbitrap™ mass spectrometer combined with a Thermo Scientific™ Trace™ 1310 GC enables high-mass-resolution measurements of molecular isotopic structure (e.g., molecular-average, position-specific, and multiple substitution measurements), but thus far has employed non-traditional, slow sample introduction methods and long integrations lasting minutes or tens of minutes to optimize measurement precision. This study examines the performance of the Orbitrap for isotope ratio measurements of analytes eluting directly from the gas chromatograph (GC) as traditional GC peaks — i.e. eluting over a period of seconds and with rapidly varying signal intensities. Such a measurement holds potential for simultaneous compound identification and isotope ratio measurement of numerous analytes separated by GC within a single acquisition. We applied this “direct elution” measurement strategy to molecular and fragment ions of para-xylene, serine, and a mixture of polycyclic aromatic hydrocarbons (PAHs) at natural isotope abundances. We built a mathematical model and used a Monte Carlo simulation to evaluate how variations in data processing decisions and GC peak characteristics (e.g., peak shape and elution timing) affect the accuracy of the resulting absolute isotope ratios and sample-standard comparisons (δ values). These case studies inform our recommendations for applying direct elution measurements. The method is appropriate for systems with large position-specific, molecular, or multiply-substituted isotopic anomalies (e.g., isotopically labelled or extraterrestrial compounds), and for compounds that produce strong molecular ions. Precisions improve when experiments are designed to (1) target ions with relatively high mass spectral intensities, (2) optimize the number of ions and range of masses admitted into the Orbitrap, and (3) minimize the nominal resolution settings while still separating relevant isobars.

Intramolecular isotope distributions, including isotope clumping and position specific fractionation, can provide proxies for the formation temperature and formation and destruction pathways of molecules. In this study, we explore the position-specific hydrogen isotope distribution in propane. We analyzed propane samples from 10 different petroleum systems with high-resolution molecular mass spectrometry. Our results show that the hydrogen isotope fractionation between central and terminal positions of natural propanes ranges from −102‰ to +205‰, a much larger range than that expected for thermodynamic equilibrium at their source and reservoir temperatures (36–63‰). Based on these findings, we propose that the hydrogen isotope structure of catagenic propane is largely controlled by irreversible processes, expressing kinetic isotope effects (KIEs). Kinetic control on hydrogen isotope composition of the products of thermal cracking is supported by a hydrous pyrolysis experiment using the Woodford Shale as substrate, in which we observed isotopic disequilibrium in the early stage of pyrolysis. We make a more general prediction of KIE signatures associated with kerogen cracking by simulating this chemistry in a kinetic Monte Carlo model for different types of kerogens. In contrast, unconventional shale fluids or hot conventional reservoirs contain propane with an isotopic structure close to equilibrium, presumably reflecting internal and/or heterogeneous exchange during high temperature storage (ca. 100–150 °C). In relatively cold (

Biogeochemical cycling of sulfur is relatively understudied in terrestrial environments compared to marine environments. However, the comparative ease of access, observation, and sampling of terrestrial settings can expand our understanding of organisms and processes important in the modern sulfur cycle. Furthermore, these sites may allow for the discovery of useful process analogs for ancient sulfur-metabolizing microbial communities at times in Earth's past when atmospheric O2 concentrations were lower and sulfide was more prevalent in Earth surface environments. We identified a new site at Santa Paula Creek (SPC) in Ventura County, CA—a remarkable freshwater, gravel-bedded mountain stream charged with a range of oxidized and reduced sulfur species and heavy hydrocarbons from the emergence of subsurface fluids within the underlying sulfur- and organic-rich Miocene-age Monterey Formation. SPC hosts a suite of morphologically distinct microbial biofacies that form in association with the naturally occurring hydrocarbon seeps and sulfur springs. We characterized the geology, stream geochemistry, and microbial facies and diversity of the Santa Paula Creek ecosystem. Using geochemical analyses and 16S rRNA gene sequencing, we found that SPC supports a dynamic sulfur cycle that is largely driven by sulfide-oxidizing microbial taxa, with contributions from smaller populations of sulfate-reducing and sulfur-disproportionating taxa. This preliminary characterization of SPC revealed an intriguing site in which to study geological and geochemical controls on microbial community composition and to expand our understanding of sulfur cycling in terrestrial environments.

Anomalous sulfur isotope compositions present in Archean rocks have been intensely scrutinized over the last 20 years because they record key aspects of Earth's atmospheric composition prior to the appearance of free molecular oxygen ca. 2.3 billion years ago. These isotopic compositions can be described as mass anomalous fractionations (MAF) and are produced in the atmosphere as UV light interacts with SO2 molecules. Most interpretations suggest that atmospheric processes generate a reduced S-phase with a positive (33S-enriched) MAF signature, as measured in pyrites, and an oxidized S-phase with a negative anomaly, as measured in bedded barite deposits. However, recent data for carbonate-associated sulfate (CAS) — a direct proxy for the isotopic composition of sulfur from seawater sulfate — in Neoarchean rocks showed no such negative values, but rather the opposite. To understand if the positive MAF anomalies we measured in Neoarchean CAS reflect secondary processes (diagenetic, metamorphic, handling) instead of original signals of Archean seawater sulfate, we collected additional sample suites with various degrees of preservation and metamorphic alteration across the Campbellrand-Malmani platform in South Africa. Results illustrate that within this comprehensive suite, less-altered samples all contain positive MAF values while secondary processes tend to either remove CAS from the sample and/or decrease the 33S-enrichment. This positive MAF signal in sulfate is therefore reasonably interpreted as a primary depositional origin, and implies that the assumption that sulfate always carries a negative MAF anomaly throughout the Archean rock record needs to be reconsidered. Our CAS observations suggest that future experiments and calculations should also consider atmospheric and/or sulfur cycling processes that can produce oxidized sulfur with a positive MAF signature.

Over the last few decades, isotopic analysis of amino acids at the compound- and position-specific levels has been rapidly advancing across diverse fields. As these techniques progress, evaluation of isotopic fractionation associated with sample workup is essential. This critical review of analytical methods through the lens of isotope geochemistry provides a benchmark for researchers across disciplines seeking to make compound- and position-specific amino acid isotope measurements. We focus on preparation, acid hydrolysis, clean-up, derivatization, separation, and C, H, N, and S isotope measurement. Despite substantial customizability across these steps, the following general recommendations should maximize recovery while minimizing isotopic fractionation. Samples should be freeze-dried and stored anoxically at ≤ –20 °C prior to conventional acid hydrolysis (6N HCl, 110 °C, 20–24 h, anoxic), which suffices for many residues. Both gas and liquid chromatographic (GC and LC, respectively) techniques are well-established and separate about 15 amino acids; LC bypasses the need for derivatization, while GC provides higher sensitivity. When derivatization is needed, n-acetyl and alkoxycarbonyl esters provide the most reproducible C isotope ratios. For compound-specific analyses, online GC–IRMS and LC–IRMS systems offer the easiest workflow, but EA–IRMS enables potential multi-element isotope analysis. Emerging techniques like high-resolution mass spectrometry are also promising for multi-element analysis and recover position-specific isotopic information. Looking forward to the next decade of innovation, isotope geochemists and ecologists can improve amino acid isotope analysis by focusing on streamlining multi-element analysis and standardizing calibration practices across laboratories.

Millennial‐scale climate anomalies punctuating the last deglaciation were expressed differently in the Northern and Southern Hemispheres. While changes in oceanic meridional overturning circulation have been invoked to explain these disparities, the nearly synchronous onset of such events requires atmospheric mediation. Yet the extent and structure of atmospheric reorganization on millennial timescales remains unclear. In particular, the role of the Southern Hemisphere westerly winds (SHWW) and associated storm tracks is poorly constrained, largely due to the paucity of accessible archives of wind behavior. Here we present a new paleohydrologic record from a Lake Hayes, New Zealand (45° S) sediment core from ~17‐9 ka. Using two independent proxies for lake hydrology (Ca/Ti in sediments and δD values of aquatic plant biomarkers), we find evidence for a wetter Antarctic Cold Reversal (ACR, 14.7‐13.0 ka) and a drying trend during the Younger Dryas (YD, 12.9‐11.6 ka) and early Holocene (11.7 ka onward in this record). Comparisons of the Lake Hayes record with other Southern Hemisphere sites indicate coherent atmospheric shifts during the ACR and YD, whereby the former is wetter/cooler and the latter is drier/warmer. The wet/cool phase is associated with a northward shift and/or strengthening of the SHWW, whereas the drier/warmer phase indicates weaker mid‐latitude winds. These climatic trends are opposite to the Northern Hemisphere. There is a decoupling of climatic trends between Southern Hemisphere low‐ and mid‐latitude climates in the early Holocene, which could be explained by several mechanisms, such as the retreat of Antarctic sea ice.

Ventilation is of primary concern for maintaining healthy indoor air quality and reducing the spread of airborne infectious disease, including COVID-19. In addition to building-level guidelines, increased attention is being placed on room-level ventilation. However, for many universities and schools, ventilation data on a room-by-room basis are not available for classrooms and other key spaces. We present an overview of approaches for measuring ventilation along with their advantages and disadvantages. We also present data from recent case studies for a variety of institutions across the United States, with various building ages, types, locations, and climates, highlighting their commonalities and differences, and examples of the use of this data to support decision making.

Mono Lake is a closed‐basin, hypersaline, alkaline lake located in Eastern Sierra Nevada, California, that is dominated by microbial life. This unique ecosystem offers a natural laboratory for probing microbial community responses to environmental change. In 2017, a heavy snowpack and subsequent runoff led Mono Lake to transition from annually mixed (monomictic) to indefinitely stratified (meromictic). We followed microbial succession during this limnological shift, establishing a two‐year (2017–2018) water‐column time series of geochemical and microbiological data. Following meromictic conditions, anoxia persisted below the chemocline and reduced compounds such as sulfide and ammonium increased in concentration from near 0 to ~400 and ~150 µM, respectively, throughout 2018. We observed significant microbial succession, with trends varying by water depth. In the epilimnion (above the chemocline), aerobic heterotrophs were displaced by phototrophic genera when a large bloom of cyanobacteria appeared in fall 2018. Bacteria in the hypolimnion (below the chemocline) had a delayed, but systematic, response reflecting colonization by sediment “seed bank” communities. Phototrophic sulfide‐oxidizing bacteria appeared first in summer 2017, followed by microbes associated with anaerobic fermentation in spring 2018, and eventually sulfate‐reducing taxa by fall 2018. This slow shift indicated that multi‐year meromixis was required to establish a sulfate‐reducing community in Mono Lake, although sulfide oxidizers thrive throughout mixing regimes. The abundant green alga Picocystis remained the dominant primary producer during the meromixis event, abundant throughout the water column including in the hypolimnion despite the absence of light and prevalence of sulfide. Our study adds to the growing literature describing microbial resistance and resilience during lake mixing events related to climatic events and environmental change.

Methane clumped isotope analysis is a tool used to constrain the formation or equilibration temperatures of methane, or to differentiate methane of thermogenic, microbial or ‘abiotic’ origins. Geothermometry applications are based on the temperature dependence of relative abundances of multiply-substituted isotopologues in thermodynamic equilibrium, whereas assignments of biogenicity or ‘abiogenicity’ rely on kinetic isotope effects associated with synthesis, which disturb clumped isotope abundances away from expected equilibrium proportions. However, kinetic processes in thermogenesis or during post-generation storage of thermogenic gas may cause isotopic disequilibrium, confounding thermometry applications or leading to ‘false positive’ identifications of microbial or abiogenic gases. Non-equilibrated clumped isotope compositions have been observed in thermogenic gases including unconventional oil-associated gases and from coal pyrolysis experiments. The isotopic disequilibria might be caused by kinetic isotope effects expressed during gas migration (including extraction), or by irreversible chemical processes, such as breaking carbon–carbon bonds in an alkyl precursor. In this study, we performed controlled pyrolysis experiments at 400 °C on n-octadecane (C18H38). We characterized the gas chemistry, and compound-specific carbon and hydrogen isotope and methane clumped isotope compositions of the gas products. We found that Δ13CH3D values (anomalies relative to a stochastic distribution of isotopes) appear to be relatively close to equilibrium at the experimental temperature, whereas Δ12CH2D2 values are 30–40‰ lower than expected for equilibrium. The large deficit in Δ12CH2D2 can be explained by assembling hydrogen atoms affected by two distinct kinetic isotope effects into a methane molecule, previously referred to as a ‘combinatorial effect’. We present a kinetic model that describes the full isotopic systematics, including anomalous Δ12CH2D2 deficits, of pyrolysis product methane. Finally, we propose a model for the isotope signatures of natural thermogenic methane where the non-equilibrium Δ12CH2D2 composition is a signature of the onset of catagenetic methane production. Our model also describes ways in which this signature disappears as further maturation drives Δ12CH2D2 to equilibrium through hydrogen exchange. Our findings demonstrate that anomalous depletion in Δ12CH2D2 is not a unique signature for microbial or putative abiotic methane, and specifically, it can be generated during pyrolytic chemistry.

The hydrogen-isotopic compositions ((2)H/(1)H ratios) of lipids in microbial heterotrophs are known to vary enormously, by at least 40% (400‰) relative. This is particularly surprising, given that most C-bound H in their lipids appear to derive from the growth medium water, rather than from organic substrates, implying that the isotopic fractionation between lipids and water is itself highly variable. Changes in the lipid/water fractionation are also strongly correlated with the type of energy metabolism operating in the host. Because lipids are well preserved in the geologic record, there is thus significant potential for using lipid (2)H/(1)H ratios to decipher the metabolism of uncultured microorganisms in both modern and ancient ecosystems. But despite over a decade of research, the precise mechanisms underlying this isotopic variability remain unclear. Differences in the kinetic isotope effects (KIEs) accompanying NADP(+) reduction by dehydrogenases and transhydrogenases have been hypothesized as a plausible mechanism. However, this relationship has been difficult to prove because multiple oxidoreductases affect the NADPH pool simultaneously. Here, we cultured five diverse aerobic heterotrophs, plus five Escherichia coli mutants, and used metabolic flux analysis to show that (2)H/(1)H fractionations are highly correlated with fluxes through NADP(+)-reducing and NADPH-balancing reactions. Mass-balance calculations indicate that the full range of (2)H/(1)H variability in the investigated organisms can be quantitatively explained by varying fluxes, i.e., with constant KIEs for each involved oxidoreductase across all species. This proves that lipid (2)H/(1)H ratios of heterotrophic microbes are quantitatively related to central metabolism and provides a foundation for interpreting (2)H/(1)H ratios of environmental lipids and sedimentary hydrocarbons.

Sulfide (H_2S, HS^−, and S^(2−)) is ubiquitous in marine porewaters as a result of microbial sulfate reduction, constituting the reductive end of the biogeochemical sulfur cycle. Stable isotopes have been widely used to constrain the sulfur cycle, because the redox transformations of sulfur compounds, such as microbial sulfate reduction, often exhibit sizable kinetic isotope effects. In contrast to sulfate ion (SO_4^(2−)), the most abundant form of dissolved sulfur in seawater, H2S is volatile and also deprotonated at near neutral pH. Equilibrium isotope partitioning between sulfide species can therefore overlap with kinetic isotope effects during reactions involving sulfide as either reactant or intermediate. Previous experimental attempts to measure equilibrium fractionation between H_2S and HS− have reached differing results, likely due to solutions of widely varying ionic strength. In this study, we measured the sulfur isotope fractionation between total dissolved sulfide and gaseous H2S at 20.6 ± 0.5 °C over the pH range from 2 to 8, and calculated the equilibrium isotope effects associated with deprotonation of dissolved H_2S. By using dilute solutions of Na2S, made possible by the improved sensitivity of mass spectrometric techniques, uncertainty in the first dissociation constant of H2S due to ionic strength could be better controlled. This in turn allowed us to close sulfur isotope mass balance for our experiments and increase the accuracy of the estimated fractionation factor. At equilibrium, aqueous H2S was enriched in ^(34)S by 0.7‰ and 3.1‰ relative to gaseous H_2S and aqueous HS−, respectively. The estimated fractionation between aqueous H_2S and HS^− lies between two earlier experimental reports, but agrees within the uncertainty of the measurements with a recent theoretical calculation.

Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved., Changes in S-isotope ratios over time provide clues to understanding the co-evolution of Earth and its biosphere. Here the authors determine the isotope effect of the first reductive enzyme in the sulfate respiration pathway and reinterpret sedimentary S-isotope records based on this biochemical constraint.

Rationale: Sulfur isotope analysis of organic sulfur‐containing molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural‐abundance sulfur isotopic compositions of the sulfur‐containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields. Methods: Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reversed‐phase high‐performance liquid chromatography (HPLC) and verified via offline liquid chromatography/mass spectrometry (LC/MS). The sulfur isotope ratios (δ³⁴S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature‐ramped chromatographic column and programmable helium carrier flow rates. Results: On‐column focusing of SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1–0.3‰ 1 s.d.) δ³⁴S measurements of 1 to 10 μg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof‐of‐concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ³⁴S values of cysteine and methionine that can be connected to biosynthetic pathways. Conclusions: We have developed a sensitive, precise method for measuring the natural‐abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment, to fundamental aspects of metabolism and biosynthesis.

Meteorites contain prebiotic, bio-relevant organic compounds including amino acids. Their syntheses could result from diverse sources and mechanisms and provide a window on the conditions and materials present in the early solar system. Here we constrain alanine’s synthetic history in the Murchison meteorite using site-specific 13C/12C measurements, reported relative to the VPDB standard. The δ13CVPDB values of −29 ± 10‰, 142 ± 20‰, and −36 ± 20‰ for the carboxyl, amine-bound, and methyl carbons, respectively, are consistent with Strecker synthesis of interstellar-medium-derived aldehydes, ammonia, and low-δ13C nebular or interstellar-medium-derived CN. We report experimentally measured isotope effects associated with Strecker synthesis, and use them to constrain the δ13C values of the alanine precursors, which we then use to construct a model that predicts the molecular-average δ13C values of 19 other organic compounds of prebiotic significance found in Murchison if they were made by our proposed synthetic network. Most of these predictions agree with previous measurements, suggesting that interstellar-medium-derived aldehydes and nebular and/or pre-solar CN could have served as substrates for synthesis of a wide range of prebiotic compounds in the early solar system.

The release of long‐stored carbon from thawed permafrost could fuel increased methanogenesis in northern lakes, but it remains unclear whether old carbon substrates released from permafrost are metabolized as rapidly by methanogenic microbial communities as recently produced organic carbon. Here, we apply methane (CH₄) clumped isotope (Δ₁₈) and ¹⁴C measurements to test whether rates of methanogenesis are related to carbon substrate age. Results from culture experiments indicate that Δ₁₈ values are negatively correlated with CH₄ production rate. Measurements of ebullition samples from thermokarst lakes in Alaska and glacial lakes in Sweden indicate strong negative correlations between CH₄ Δ₁₈ and the fraction modern carbon. These correlations imply that CH₄ derived from older carbon substrates is produced relatively slowly. Relative rates of methanogenesis, as inferred from Δ₁₈ values, are not positively correlated with CH₄ flux estimates, highlighting the likely importance of environmental variables other than CH₄ production rates in controlling ebullition fluxes.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1–2‰ for sulfate δ³⁴S and δ¹⁸O and nitrate δ¹⁵N and δ¹⁸O, based on in-house and international standards. Less abundant variants such as δ¹⁷O, δ³³S, and δ³⁶S, and the ³⁴S–¹⁸O “clumped” sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine ¹⁸O/³⁴S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

The Last Glacial-Interglacial Transition (LGIT) was a dynamic stage of Earth's history, and the difficulty of reconstructing this complex interval may be compounded by divergent proxy records, often collected from the same archive. To overcome this obstacle, we exploit the contrasting biological preferences of chironomids to both summer temperature and degree days (positive) and pollen to both the length of the growing season (positive) and winter duration (negative), recorded by a small lake in the central North Island of New Zealand. The climate proxy records are anchored to shifting zonal boundaries (e.g., southern westerly wind belt) via the hydrogen isotope ratios of leaf wax n-alkanes (δDwax). These results enable us to interrogate the structure of the LGIT and address two fundamental questions: 1) Is there evidence for Holocene-like temperatures during the early deglacial? and 2) Were early Holocene summers cool? Lake sediment δDwax values indicate a poleward retreat of the westerlies around 18,000 calibrated years before present (cal kyr BP), signalling the onset of climate amelioration for the region. Remarkably, the independently derived summer and mean annual temperature reconstructions are anti-phased. Chironomid-inferred summer temperatures surpass modern values by 17.5 cal kyr BP, whereas pollen-inferred mean annual temperatures remain supressed. Summers cool from 17.5 to 11 cal kyr BP, when they reach a minimum for the record, while winter and mean annual temperatures simultaneously warm to a maximum. The chironomid record generally traces regional insolation, although an Antarctic template imprints on this trend with declining summer temperatures during the Antarctic Cold Reversal (ACR) and warming during the Younger Dryas (YD). Forest development during the early LGIT, on the other hand, is supressed by cool, dry winters, punctuated by severe frosts, despite being set against a backdrop of overall warming; the successional pattern is best explained by a latitudinal retreat of the westerlies. Our findings underscore the complementarity of multiple bioproxy responses and reveal the importance of seasonal heat and energy distribution during the LGIT.

Microbial sulfate reduction exhibits a normal isotope effect, leaving unreacted sulfate enriched in 34 S and producing sulfide that is depleted in 34 S. However, the magnitude of sulfur isotope fractionation is quite variable. The resulting changes in sulfur isotope abundance have been used to trace microbial sulfate reduction in modern and ancient ecosystems, but the intracellular mechanism(s) underlying the wide range of fractionations remains unclear. Here we report the concentrations and isotopic ratios of sulfur metabolites in the dissimilatory sulfate reduction pathway of Desulfovibrio alaskensis . Intracellular sulfate and APS levels change depending on the growth phase, peaking at the end of exponential phase, while sulfite accumulates in the cell during stationary phase. During exponential growth, intracellular sulfate and APS are strongly enriched in 34 S. The fractionation between internal and external sulfate is up to 49‰, while at the same time that between external sulfate and sulfide is just a few permil. We interpret this pattern to indicate that enzymatic fractionations remain large but the net fractionation between sulfate and sulfide is muted by the closed-system limitation of intracellular sulfate. This ‘reservoir effect’ diminishes upon cessation of exponential phase growth, allowing the expression of larger net sulfur isotope fractionations. Thus, the relative rates of sulfate exchange across the membrane versus intracellular sulfate reduction should govern the overall (net) fractionation that is expressed. A strong reservoir effect due to vigorous sulfate reduction might be responsible for the well-established inverse correlation between sulfur isotope fractionation and the cell-specific rate of sulfate reduction, while at the same time intraspecies differences in sulfate uptake and/or exchange rates could account for the significant scatter in this relationship. Our approach, together with ongoing investigations of the kinetic isotope fractionation by key enzymes in the sulfate reduction pathway, should provide an empirical basis for a quantitative model relating the magnitude of microbial isotope fractionation to their environmental and physiological controls.

Rationale: Dissolved sulfur species are of significant interest, both as important substrates for microbial activities and as key intermediaries in biogeochemical cycles. Species of intermediate oxidation state such as sulfite, thiosulfate, and thiols are of particular interest but are notoriously difficult to analyze, because of low concentrations and rapid oxidation during storage and analysis. Methods: Dissolved sulfur species are reacted with monobromobimane which yields a fluorescent bimane derivative that is stable to oxidation. Separation by Ultra-Performance Liquid Chromatography (UPLC) on a C18 column yields baseline resolution of analytes in under 5 minutes. Fluorescence detection (380 nm excitation, 480 nm emission) provides highly selective and sensitive quantitation, and Time of Flight Mass Spectrometry (TOF-MS) is used to quantify isotopic abundance, providing the ability to detect stable isotope tracers (either ^(33)S or ^(34)S). Results: Sulfite, thiosulfate, methanethiol, and bisulfide were quantified with on-column detection limits of picomoles (μM concentrations). Other sulfur species with unshared electrons are also amenable to analysis. TOF-MS detection of ^(34)S enrichment was accurate and precise to within 0.6% (relative) when sample and standard had similar isotope ratios, and was able to detect enrichments as small as 0.01 atom%. Accuracy was validated by comparison to isotope-ratio mass spectrometry. Four example applications are provided to demonstrate the utility of this method. Conclusions: Derivatization of aqueous sulfur species with bromobimane is easily accomplished in the field, and protects analytes from oxidation during storage. UPLC separation with fluorescence detection provides low μM detection limits. Using a high-resolution TOF-MS, accurate detection of as little as 0.01% ^(34)S label incorporation into multiple species is feasible. This provides a useful new analytical window into microbial sulfur cycling.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effect of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. Microbial community composition was measured by NanoString DNA analysis and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two different anteiso fatty acids (a-C15:0 and a-C17:0) using gas chromatography–mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae and hence their isotopic enrichment was interpreted as a growth proxy for S. aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients, thus deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small non-microbial background source detected in some patient samples, a-C15:0 fatty acid still appear to be a relatively robust proxy for CF pathogens, revealing a median generation time of ~1.5 days, similar to prior observations. Contrary to our expectations, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two best practices for application of stable isotope probing in CF sputum: (1) parallel determination of microbial community composition in CF sputum using culture-independent tools, and (2) analysis of samples with a minimum a-C15:0 concentration of 0.1 weight percent of saturated fatty acids.IMPORTANCEIn chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop an new workflow for the application of stable isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

Many previous studies have examined abundances of deuterium (D) and ^(13)C within small organic molecules. Recent advances in analytical instrumentation add the abilities to measure site-specific and multiply substituted isotopologues of natural organics. Here we perform first-principles calculations of the equilibrium distributions of ^(13)C and D in the volatile alkanes (including both single and multiple substitutions), as a guide to the interpretation of current measurements and as a basis for anticipating isotope effects that might be examined with future analytical techniques. The models we present illustrate several common themes of the isotopic structures of the small alkanes, including; temperature dependent enrichment of clumped isotope species, with amplitudes in the order D–D > ^(13)C–D > ^(13)C–^(13)C; similarity in strength of such clumped isotope effects between different molecules (e.g., ^(13)C–D clumping is ∼5‰ enriched at 300 K in methane, ethane and propane); a ∼10× contrast between the amplitudes of stronger adjacent substitution of two heavy isotopes vs. weaker non-adjacent substitution; temperature-dependent site-specific fractionation of D and ^(13)C into interior positions of molecules relative to terminal methyl groups; and a relatively simple additive effect to the overall amplitude of enrichment when clumped and site specific effects combine in the same isotopologue. We suggest that the most promising tools suggested by our results are isotopic thermometers based on site-specific distribution of deuterium, which exhibits strong (∼100‰), highly temperature dependent fractionation between methyl groups and methylene carbon positions in propane (and likely other larger n-alkanes).

The sulfur isotopic fractionation associated with the formation of organic sulfur compounds (OSCs) during thermochemical sulfate reduction (TSR) was studied using gold-tube pyrolysis experiments to simulate TSR. The reactants used included n-hexadecane (n-C_(16)) as a model organic compound with sulfate, sulfite, or elemental sulfur as the sulfur source. At the end of each experiment, the S-isotopic composition and concentration of remaining sulfate, H_2S, benzothiophene, dibenzothiophene, and 2-phenylthiophene (PT) were measured. The observed S-isotopic fractionations between sulfate and BT, DBT, and H_2S in experimental simulations of TSR correlate well with a multi-stage model of the overall TSR process. Large kinetic isotope fractionations occur during the first, uncatalyzed stage of TSR, 12.4‰ for H_2S and as much as 22.2‰ for BT. The fractionations decrease as the H_2S concentration increases and the reaction enters the second, catalyzed stage. Once all of the oxidizable hydrocarbons have been consumed, sulfate reduction ceases and equilibrium partitioning then dictates the fractionation between H_2S and sulfate (∼17‰). Experiments involving sparingly soluble CaSO_4 show that during the second catalytic phase of TSR the rate of sulfate reduction exceeds that of sulfate dissolution. In this case, there is no apparent isotopic fractionation between source sulfate and generated H_2S, as all of the available sulfate is effectively reduced at all reaction times. When CaSO_4 is replaced with fully soluble Na_2SO_4, sulfate dissolution is no longer rate limiting and significant S-isotopic fractionation is observed. This supports the notion that CaSO_4 dissolution can lead to the apparent lack of fractionation between H_2S and sulfate produced by TSR in nature. The S-isotopic composition of individual OSCs record information related to geochemical reactions that cannot be discerned from the δ^(34)S values obtained from bulk phases such as H2S, oil, and sulfate minerals, and provide important mechanistic details about the overall TSR process.

The hydrogen isotopic compositions of sedimentary hydrocarbon molecules are now being used to address a range of scientific questions, from paleoclimate to environmental reconstruction to understanding of petroleum systems. Here I review the environmental, biological, and physical/chemical factors that influence the H isotopic compositions of sedimentary hydrocarbons. A hierarchy of four main controls can be recognized: i) the composition of environmental water that serves as ultimate hydrogen source to the biosphere ii) physiologic and metabolic processes in organisms that fix water hydrogen into organic molecules, iii) hydrogen exchange processes that alter D/H ratios slowly over time under geologic conditions, and iv) kinetic fractionations that arise during the thermal conversion of sedimentary organic matter to liquid hydrocarbons. Variations in the terrestrial hydrologic cycle, and in biologic fractionations, create lipids with δD values spanning a huge range, and carrying abundant information about source organisms and environments. This information is gradually lost over geologic timescales due to hydrogen exchange, although the rates of this process appear to vary by orders of magnitude in clastic versus carbonate sediments. The H isotopes of hydrocarbons in many sediments of low to moderate thermal maturity may only be partially exchanged. Finally, additional kinetic fractionations are imposed during thermal cracking to generate mobile hydrocarbons. The δD values of sedimentary hydrocarbons are thus concluded to generally reflect a complicated combination of processes. Although it is tempting to view this as a failure of the isotopic record, there are numerous opportunities for understanding environment, diagenetic, and catagenetic processes if we can learn to quantitatively disentangle the competing fractionations.

We present a Younger Dryas–Holocene record of the hydrogen isotopic composition of sedimentary plant waxes (δD_(wax)) from the southern European Alps (Lake Ghirla, N-Italy) to investigate its sensitivity to climatic forcing variations in this mid-latitude region (45°N). A modern altitudinal transect of δD values of river water and leaf waxes in the Lake Ghirla catchment is used to test present-day climate sensitivity of δD_(wax). While we find that altitudinal effects on δD_(wax) are minor at our study site, temperature, precipitation amount, and evapotranspiration all appear to influence δD_(wax) to varying extents. In the lake-sediment record, δD_(wax) values vary between −134 and −180‰ over the past 13 kyr. The long-term Holocene pattern of δD_(wax) parallels the trend of decreasing temperature and is thus likely forced by the decline of northern hemisphere summer insolation. Shorter-term fluctuations, in contrast, may reflect both temperature and moisture-source changes. During the cool Younger Dryas and Little Ice Age (LIA) periods we observe unexpectedly high δD_(wax) values relative to those before and after. We suggest that a change towards a more D-enriched moisture source is required during these intervals. In fact, a shift from northern N-Atlantic to southern N-Atlantic/western Mediterranean Sea sources would be consistent with a southward migration of the Westerlies with climate cooling. Prominent δD_(wax) fluctuations in the early and middle Holocene are negative and potentially associated with temperature declines. In the late Holocene (

RATIONALEMicrobial growth rate is an important physiological parameter that is challenging to measurein situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times ofS. aureusin cystic fibrosis (CF) infections can be determined by D2O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids.METHODSLipid extracts of D2O-treatedE. colicultures were measured on LC-ESI-MS instruments equipped with TOF and Orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio GC-MS. We then develop an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we apply D2O-labeling lipidomics to clinical samples from CF patients with chronic lung infections.RESULTSLipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In theE. colicultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific forS. aureusin CF sputum, indicates an average generation time of the pathogen on the order of one cell doubling per day.CONCLUSIONSThis study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2O.

Site-specific carbon isotope measurements of organic compounds potentially recover information that is lost in a conventional, ‘bulk' isotopic analysis. Such measurements are useful because isotopically fractionating processes may have distinct effects at different molecular sites, and thermodynamically equilibrated populations of molecules tend to concentrate heavy isotopes in one molecular site versus another. Most recent studies of site-specific ^(13)C in organics use specialized Nuclear Magnetic Resonance (NMR) techniques or complex chemical degradations prior to mass spectrometric measurements. Herein we present the first application of a new mass spectrometric technique that reconstructs the site-specific carbon isotope composition of propane based on measurements of the ^(13)C/^(12)C ratios of two or more fragment ions that sample different proportions of the terminal and central carbon sites. We apply this method to propane from laboratory experiments and natural gas samples to explore the relationships between site-specific carbon isotope composition, full-molecular δ^(13)C, thermal maturity, and variation in organic matter precursors. Our goal is to advance the understanding of the sources and histories of short-chain alkanes within geologic systems. Our findings suggest that propane varies in its site-specific carbon isotope structure, which is correlated with increasing thermal maturity, first increasing in terminal position δ^(13)C and then increasing in both center and terminal position δ^(13)C. This pattern is observed in both experimental and natural samples, and is plausibly explained by a combination of site-specific, temperature-dependent isotope effects associated with conversion of different precursor molecules (kerogen, bitumen, and/or oil) to propane, differences in site-specific isotopic contents of those precursors, and possibly distillation of reactive components of those precursors with increasing maturity. We hypothesize that the largest changes in site-specific isotopic content of propane occur when bitumen and/or oil replace kerogen as the dominant precursors. If correct, this phenomenon could have significant utility for understanding gas generation in thermogenic petroleum systems.

The carbon isotope ratio (δ^(13)C value) of marine particulates is a potentially useful tracer for elucidating pathways of carbon flow in the marine environment. Different species of phytoplankton vary in fractionation vs. CO_2 by up to 24‰ in laboratory cultures under varying nutrient and growth conditions, a signal that should propagate through the microbial food web. However, such contrasts have been difficult to confirm in field measurements due to analytical limitations. Here, we combine fluorescence-activated cell sorting (FACS) with a specialized micro-combustion interface and isotope-ratio mass spectrometry (SWiM-IRMS) to provide some of the first direct measurements of whole-cell δ^(13)C values for specific phytoplankton populations in the wild. For three samples collected off Scripps Pier in 2010–2011, Synechococcus averages δ^(13)C values of −25.7 ± 2.0‰, Prochlorococcus averages −23.0 ± 1.3, and diatoms average −20.8 ± 1.7‰. Diatoms were ∼3‰ enriched in ^(13)C when measured during a bloom (March 2011) as compared with mid-summer (July 2010). Sorted particles thought to represent living heterotrophic bacteria averaged −25.4 ± 2.5‰, whereas total filterable particles averaged −19.6 ± 1.0‰, indicating a strong similarity to diatom biomass. These variations demonstrate that in situ differences in δ^(13)C among different populations of particles can be exploited to follow carbon flow through successive trophic levels, and throughout organic matter remineralization, sinking, and preservation.

© 2015 Elsevier Ltd. Sources of methane to sedimentary environments are commonly identified and quantified using the stable isotopic compositions of methane. The methane "clumped-isotope geothermometer", based on the measurement of multiply substituted methane isotopologues (13CH3D and 12CH2D2), shows promise in adding new constraints to the sources and formational environments of both biogenic and thermogenic methane. However, questions remain about how this geothermometer behaves in systems with mixtures of biogenic and thermogenic gases and different biogenic environments. We have applied the methane clumped-isotope thermometer to a mixed biogenic-thermogenic system (Antrim Shale, USA) and to biogenic gas from gas seeps (Santa Barbara and Santa Monica Basin, USA), a pond on the Caltech campus, and methanogens grown in pure culture. We demonstrate that clumped-isotope based temperatures add new quantitative constraints to the relative amounts of biogenic vs. thermogenic gases in the Antrim Shale indicating a larger proportion (~50%) of thermogenic gas in the system than previously thought. Additionally, we find that the clumped-isotope temperature of biogenic methane appears related to the environmental settings in which the gas forms. In systems where methane generation rates appear to be slow (e.g., the Antrim Shale and gas seeps), microbial methane forms in or near both internal isotopic equilibrium and hydrogen-isotope equilibrium with environmental waters. In systems where methane forms rapidly, microbial methane is neither in internal isotopic equilibrium nor hydrogen-isotope equilibrium with environmental waters. A quantitative model of microbial methanogenesis that incorporates isotopes is proposed to explain these results.

The sedimentary record of molecular fossils (biomarkers) can potentially provide important insights into the composition of ancient organisms; however, it only captures a small portion of their original lipid content. To interpret what remains, it is important to consider the potential for functional overlap between different lipids in living cells, and how the presence of one type might impact the abundance of another. Hopanoids are a diverse class of steroid analogs made by bacteria and found in soils, sediments, and sedimentary rocks. Here, we examine the trade-off between hopanoid production and that of other membrane lipids. We compare lipidomes of the metabolically versatile α-proteobacterium Rhodopseudomonas palustris TIE-1 and two hopanoid mutants, detecting native hopanoids simultaneously with other types of polar lipids by electrospray ionization mass spectrometry. In all strains, the phospholipids contain high levels of unsaturated fatty acids (often >80 %). The degree to which unsaturated fatty acids are modified to cyclopropyl fatty acids varies by phospholipid class. Deletion of the capacity for hopanoid production is accompanied by substantive changes to the lipidome, including a several-fold rise of cardiolipins. Deletion of the ability to make methylated hopanoids has a more subtle effect; however, under photoautotrophic growth conditions, tetrahymanols are upregulated twofold. Together, these results illustrate that the ‘lipid fingerprint’ produced by a micro-organism can vary depending on the growth condition or loss of single genes, reminding us that the absence of a biomarker does not necessarily imply the absence of a particular source organism.

s and papers in preparation describe the ‘‘tentative’’ detection of dichloropropane and chlorobenzene at levels of a few tens of nanograms per gram in a core drilled into a mudstone at Yellowknife Bay (e.g.. Summons et al., 2014a,b). A significant caveat here is that all three missions used thermolysis and pyrolysis to volatilize organic compounds so that they are amenable to gas chromatographic and mass spectrometric analysis. A series of studies (Navarro-Gonzalez et al., 2006, 2010, 2011; NavarroGonzalez and McKay, 2011) suggested that the presence of perchlorate and other oxidants in the martian regolith renders the Viking results difficult to interpret because of the high probability of either oxidizing or chlorinating indigenous organic molecules during heating. These observations have been contested (Biemann, 2007). As a result of these complications, the Panel did not rely on the earlier non-detections of organics in Mars regolith. The more recent detections of chlorobenzene (or possibly its aromatic precursors) and other chlorohydrocarbons by MSL (e.g., Freissinet et al., 2014) are considered to provide likely lower limits for these compounds. The OCP briefly considered organic concentrations in analog terrestrial rocks as another constraint on what to expect on Mars. Fine-grained sedimentary rocks that have not been oxidized or weathered constitute the type of sample that we might hope to return from Mars. On Earth, similar rocks commonly contain > 0.1% (100 ppm) TOC, and contain many individual biomarkers at levels > 1 ppm. Even those sediments considered to be relatively poor in organics contain > 0.01% (10 ppm) TOC (Mayer, 1994), and yield individual biomarkers at levels > 10 ng/g (e.g., Lipp et al., 2008). Soils from the Atacama Desert are reported to have 32 ppm TOC (Navarro-Gonzalez et al., 2010), an order of magnitude below typical ‘‘organic poor’’ marine sediments. Subcritical water extraction of subsurface Atacama soils ( Jungay region) followed by derivatization and capillary electrophoresis of the fluorescently labeled amines has demonstrated individual amines and amino acids at the 50– 100 ng/g level (Skelley et al., 2007). The earlier MSR SSG II panel considered a more extreme example of organic-poor sediments (i.e., a highly oxidized sedimentary rock that had been buried and undergone thermal maturation). Although such rocks have similar levels of TOC (approximately 0.01%), diagenesis has rendered most biomarkers into macromolecular kerogen, which is not extractable. They thus estimated expected concentrations for hydrocarbon biomarkers of 0.1–1.0 ng/g in such a rock (see Section 2.4 for details of this calculation). There are several limitations to using such terrestrial analogs to predict concentrations of martian organics. First and foremost, there is no a priori reason to expect that concentrations of organics in terrestrial rocks would be indicative of those on Mars. Indeed, valid arguments can be made for their being either higher or lower than indigenous martian concentrations. For example, with lower organic input and more oxidizing subsurface conditions, martian rocks might have lower organic concentrations. If microbial activity were more limited (or absent) on Mars, residual organic concentrations might be higher. Second, it is unclear which terrestrial rocks, sediments, or soils we should choose as appropriate analogs. Even considering that oxidized rocks represent a reasonable lower bound for those found on Mars, the goal of the mission is clearly not to sample and return the most oxidized martian rocks. Indeed, it is unclear whether scientific goals could be met with such a rock even given zero organic contamination. The third concern expressed by the OCP is that hydrocarbons (the dominant biomarkers in thermally mature terrestrial rocks, and those considered primarily by the MSR SSG II report) may not be the class of organics that are most abundant or interesting on Mars. With no active tectonics to deeply bury sediments under reducing conditions, biomolecules (or even meteoritic organic compounds) might be transformed to more oxidized species rather than more reduced ones. In summary, consideration of terrestrial analog rocks indicates that organic concentrations in martian rocks might span a huge range around those directly measured in meteorites. We therefore conclude that this line of argument provides little firm footing on which to construct quantitative limits. A final constraint on expected concentrations in the absence of martian biota, previously considered by the OCSSG report and by Benner et al. (2000), can be derived from estimated rates of delivery of organic carbon to the surface of Mars by meteorites. Meteorites deposit an estimated 2.4 · 10 g/year of organic carbon to the martian surface (Flynn, 1996). If allowed to accumulate over 3 billion years, and given a Martian surface area of 3.6 · 10 m, this would result in 20 kg/m of organic carbon. Assuming a mixing depth of 1 m and rock density of 4 g/mL results in a predicted TOC concentration of 5 mg/g. A much more conservative mixing depth of 100 m would lower this to 50 lg/g. If we presume that this organic carbon has a molecular makeup similar to that of Murchison (see Table 7), where functional classes of molecules represent approximately 0.05% of TOC, we predict approximately 2.5 lg/g of each class of organics (1 m mixing depth). Further assuming that each class comprises 10–100 compounds, this yields a final prediction of approximately 20–200 ng/g per compound (or 0.2–2 ng/g for the 100-m mixing depth). These estimates span Table 7. Distribution of Carbon in the Murchison CM2 Meteorite

© 2017 Elsevier Ltd The isotopic composition of methane is of longstanding geochemical interest, with important implications for understanding petroleum systems, atmospheric greenhouse gas concentrations, the global carbon cycle, and life in extreme environments. Recent analytical developments focusing on multiply substituted isotopologues (‘clumped isotopes’) are opening a valuable new window into methane geochemistry. When methane forms in internal isotopic equilibrium, clumped isotopes can provide a direct record of formation temperature, making this property particularly valuable for identifying different methane origins. However, it has also become clear that in certain settings methane clumped isotope measurements record kinetic rather than equilibrium isotope effects. Here we present a substantially expanded dataset of methane clumped isotope analyses, and provide a synthesis of the current interpretive framework for this parameter. In general, clumped isotope measurements indicate plausible formation temperatures for abiotic, thermogenic, and microbial methane in many geological environments, which is encouraging for the further development of this measurement as a geothermometer, and as a tracer for the source of natural gas reservoirs and emissions. We also highlight, however, instances where clumped isotope derived temperatures are higher than expected, and discuss possible factors that could distort equilibrium formation temperature signals. In microbial methane from freshwater ecosystems, in particular, clumped isotope values appear to be controlled by kinetic effects, and may ultimately be useful to study methanogen metabolism.

Rationale: Methylation protocols commonly call for acidic, hot conditions that are known to promote organic ^1H/^2H exchange in aromatic and aliphatic C—H bonds. Here we tested two such commonly-used methods and compared a third that avoids these acidic conditions, to quantify isotope effects with each method and to directly determine acidic-exchange rates relevant to experimental conditions. Methods: We compared acidic and non-acidic methylation approaches catalyzed by hydrochloric acid, acetyl chloride and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / DMAP (4-dimethylaminopyridine) respectively. These were applied to two analytes: phthalic acid (an aromatic) and octacosanoic acid (an aliphatic). We analyzed yield by gas chromatography flame ionization (GC/FID) and hydrogen and carbon isotopic composition by isotope ratio mass spectrometry (GC/IRMS). We quantified the ^1H/^2H exchange rate on dimethyl phthalate under acidic conditions with proton nuclear magnetic resonance (^1H-NMR) measurements. Results: The δ^2H and δ^(13)C values and yield were equivalent among the three methods for methyl octacosanoate. The two acidic methods resulted in comparable yield and isotopic composition of dimethyl phthalate; however, the non-acidic method resulted in lower δ^2H and δ^(13)C values perhaps due to low yields. Concerns over acid-catalyzed ^1H/^2H exchange are unwarranted as the effect was trivial over a 12-hour reaction time. Conclusions: We find product isolation yield and evaporation to be the main concerns in the accurate determination of isotopic composition. ^1H/^2H exchange reactions are too slow to cause measurable isotope fractionation over the typical duration and reaction conditions used in methylation. Thus, we are able to recommend continued use of acidic catalysts in such methylation reactions for both aliphatic and aromatic compounds.