Accuracy and Practical Considerations for Doubly Labeled Water Analysis in Nutrition Studies Using a Laser-Based Isotope Instrument (Off-Axis Integrated Cavity Output Spectroscopy).

BACKGROUND: Given the utility of the doubly labeled water (DLW) method for determination of energy expenditure, additional techniques for isotope analysis of the samples are welcome. Laser-based instruments are one such new analytical tool, but their accuracy and feasibility for DLW studies are grossly understudied. OBJECTIVES: We assessed the accuracy of laser-based isotope ratio measurements as part of the DLW method for estimation of carbon dioxide production rate (rCO2) and total energy expenditure (TEE), in between-group comparison study designs. METHODS: Urine samples from a previous study were analyzed with a laser-based instrument [off-axis integrated cavity output spectroscopy (OA-ICOS)]. In that study, participants consumed a high-, moderate-, or low-carbohydrate diet for 20 wk; urine samples were obtained in weeks 18-20 before and after a 2H- and 18O-enriched water dose. Isotope ratios (δ2H and δ18O), rCO2, and TEE calculated by standard methods were compared to results previously obtained with the standard technique of isotope ratio mass spectrometry (IRMS). Bias, SD, and bias ± 1.96SD bands between IRMS and OA-ICOS were computed. RESULTS: The between OA-ICOS and IRMS rCO2 and TEE trends were equivalent (within 1.2% and 4.1%, respectively), in spite of the differences in measured δ18O values at high enrichment levels. The OA-ICOS δ18O values displayed an increasing offset from the IRMS results as the 18O enrichment increased (mean ± SD 4.6-5.7‰ ± 2‰ offset at the time point with highest 18O enrichment, ∼135‰), whereas the hydrogen isotope ratio (δ2H) differed only slightly between the methods (mean offset -4.9‰ for all time points). The between-diet differences in TEE from the previous study were recapitulated with a smaller subset of participants and time points. CONCLUSIONS: OA-ICOS analysis is an accurate and feasible technique for the DLW method. Given the δ18O offset observed at high enrichment, validation of each OA-ICOS instrumental setup against established methods (e.g., IRMS) is recommended.

Carbonate-hosted microbial communities are prolific and pervasive methane oxidizers at geologically diverse marine methane seep sites.

At marine methane seeps, vast quantities of methane move through the shallow subseafloor, where it is largely consumed by microbial communities. This process plays an important role in global methane dynamics, but we have yet to identify all of the methane sinks in the deep sea. Here, we conducted a continental-scale survey of seven geologically diverse seafloor seeps and found that carbonate rocks from all sites host methane-oxidizing microbial communities with substantial methanotrophic potential. In laboratory-based mesocosm incubations, chimney-like carbonates from the newly described Point Dume seep off the coast of Southern California exhibited the highest rates of anaerobic methane oxidation measured to date. After a thorough analysis of physicochemical, electrical, and biological factors, we attribute this substantial metabolic activity largely to higher cell density, mineral composition, kinetic parameters including an elevated Vmax, and the presence of specific microbial lineages. Our data also suggest that other features, such as electrical conductance, rock particle size, and microbial community alpha diversity, may influence a sample's methanotrophic potential, but these factors did not demonstrate clear patterns with respect to methane oxidation rates. Based on the apparent pervasiveness within seep carbonates of microbial communities capable of performing anaerobic oxidation of methane, as well as the frequent occurrence of carbonates at seeps, we suggest that rock-hosted methanotrophy may be an important contributor to marine methane consumption.

Growing up in Ancient Sardinia: Infant-toddler dietary changes revealed by the novel use of hydrogen isotopes (δ2H).

Detailed information about the lives and deaths of children in antiquity is often in short supply. Childhood dietary histories are, however, recorded and maintained in the teeth of both juveniles and adults. Primary tooth dentinal collagen does not turn over, preserving a sequential record of dietary changes. The use of nitrogen (δ15N) and carbon (δ13C) isotope values of incrementally sampled dentin are used in the study of breastfeeding practices but evidence for the addition of weaning foods, both in terms of mode and, particularly, duration, has remained analytically inaccessible to date. Here, we demonstrate how the novel use hydrogen isotope (δ2H) values of sequentially micro-sampled dentin collagen, measured from individuals excavated from a Punic cemetery, in Sardinia, Italy, can serve as a proxy for weaning food type and duration in ancient childhood diet. The weaning rate and age, based on the decline in δ15N and δ13C values of permanent first molars and the concomitant increase in δ2H, appears to be broadly similar among six individuals. Hydrogen isotopes vary systematically from a low value soon after birth, rising through early childhood. The early post-birth values can be explained by the influence of 2H-depleted lipids from mother's breastmilk and the later δ2H rise is consistent with, among other things, a substantial portion of boiled foodstuffs, such as the higher δ2H values observed in porridge. Overall δ2H in dentin shows great promise to elucidate infant and childhood feeding practices, and especially the introduction of supplementary foods during the weaning process.

Mediterranean precipitation isoscape preserved in bone collagen δ2H.

The prehistory of the Mediterranean region has long been a subject of considerable interest, particularly the links between human groups and regions of origin. We utilize the spatial variation in the δ2H and δ18O values of precipitation (isoscapes) to develop proxies for geographic locations of fauna and humans. Bone collagen hydrogen isotope ratios (δ2H) in cattle (and to a lesser extent, ovicaprids) across the Mediterranean reflect the isotopic differences observed in rainfall (but δ18O values do not). We conclude that δ2H in herbivore bone collagen can be used as a geolocation tracer and for palaeoenvironmental studies such as tracing past isotopic variations in the global hydrological cycle. In contrast, human bone δ2H values are relatively tightly grouped and highly distinct from precipitation δ2H values, likely due to human-specific food practices and environmental modifications. Given the inter-species variability in δ2H, care should be taken in the species selected for study.

The interconversion of δ2 H values of collagen between thermal conversion reactor configurations.

RATIONALE: Different thermal conversion reactor packings result in distinct δ2 H values in nitrogen-containing materials, such as bone collagen. An older 'traditional' glassy carbon packing method causes incomplete conversion of N-containing samples into H2 gas, resulting in altered δ2 H values compared with the complete conversion of hydrogen obtained with a chromium-packed reactor. Given that δ2 H values from collagen are gaining importance in palaeoecological and archaeological studies, a determination of the relationship between δ2 H values produced with a glassy-carbon-packed and a chromium-packed reactor is needed. METHODS: We obtained δ2 H values (normalized on the VSMOW-SLAP scale) from both glassy-carbon-packed (GP) and chromium-packed (Cr) reactor configurations from bone collagen (n = 231) from a variety of archaeological sites, using a High-Temperature Conversion Elemental Analyzer (TC/EA) coupled to a Delta Plus XP isotope ratio mass spectrometer. RESULTS: δ2 H values from both methods are linearly correlated (r2  = 0.934) and yield the following interconversion equation, δ2 H(Cr) = 1.054 δ2 H(GP) + 11.6‰ (95% conf. slope 1.020-1.090, intercept 10.6-12.6), and a mean difference of δ2 H(Cr) - Î´2 H(GP) = 10.1‰ (1 sd 5.2, 1 se 0.3, n = 231). CONCLUSIONS: We recommend adopting this interconversion between δ2 H values produced with a glassy-carbon-packed and chromium-packed reactor for bone collagen only, with appropriate propagation of uncertainty.

Harnessing a methane-fueled, sediment-free mixed microbial community for utilization of distributed sources of natural gas.

Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy-rich, yet the most efficient methane-activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by-products at a comparable rate and in near-stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost-effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep-sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep-sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full-scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane-oxidizing, sulfide-generating mesocosm incubations. Metabolic activity required >∼40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane-dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth-based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane-activating microbial communities using a low-mass and sediment-free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use.

Monodeuterated Methane, an Isotopic Tool To Assess Biological Methane Metabolism Rates.

Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring these metabolic rates presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for investigating microbial methane metabolism based on hydrogen atom dynamics, which is complementary to carbon-focused assessments of methanotrophy. The method uses monodeuterated methane (CH3D) as a metabolic substrate, quantifying the aqueous D/H ratio over time using off-axis integrated cavity output spectroscopy. This approach represents a nontoxic, comparatively rapid, and straightforward approach that supplements existing radiotopic and stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional dimension for examining rates and pathways of methane metabolism. We provide direct comparisons between the CH3D procedure and the well-established 14CH4 radiotracer method for several methanotrophic systems, including type I and II aerobic methanotroph cultures and methane-seep sediment slurries and carbonate rocks under anoxic and oxic incubation conditions. In all applications tested, methane consumption values calculated via the CH3D method were directly and consistently proportional to 14C radiolabel-derived methane oxidation rates. We also employed this method in a nontraditional experimental setup, using flexible, gas-impermeable bags to investigate the role of pressure on seep sediment methane oxidation rates. Results revealed an 80% increase over atmospheric pressure in methanotrophic rates the equivalent of ~900-m water depth, highlighting the importance of this parameter on methane metabolism and exhibiting the flexibility of the newly described method. IMPORTANCE Microbial methane consumption is a critical component of the global carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon exploitation. Nonetheless, quantifying methane metabolism typically involves logistically challenging methods and/or specialized equipment; these impediments have limited our understanding of methane fluxes and reservoirs in natural systems, making effective management difficult. Here, we offer an easily implementable, precise method using monodeuterated methane (CH3D) that advances three specific aims. First, it allows users to directly compare methane consumption rates between different experimental treatments of the same inoculum. Second, by empirically linking the CH3D procedure with the well-established 14C radiocarbon approach, we determine absolute scaling factors that facilitate rate measurements for several aerobic and anaerobic systems of interest. Third, CH3D represents a helpful tool in evaluating the relationship between methane activation and full oxidation in methanotrophic metabolisms. The procedural advantages, consistency, and novel research questions enabled by the CH3D method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.

Hydrogen isotopic analysis with a chromium-packed reactor of organic compounds of relevance to ecological, archaeological, and forensic applications.

RATIONALE: The δ(2) H values of some nitrogen-containing organic compounds measured by High-Temperature Conversion (HTC) with a glassy carbon reactor have been shown to be inaccurate. A probable explanation for these analytical inaccuracies is the formation of HCN, allowing some hydrogen atoms to escape isotope ratio measurement. We assess this isotopic effect in sample types commonly used for (paleo)ecological, environmental, archaeological, and forensic investigations. METHODS: The δ(2) HVSMOW-SLAP values and mass fraction H using a factory-recommended glassy carbon HTC reactor packing were compared with those obtained from using two Cr-containing reactor packings for a variety of N-containing substances, including amino acids, collagen, hair, and silk. RESULTS: δ(2) HVSMOW-SLAP values and mass fraction H differed by reactor packing for most, but not all, N-containing samples. The δ(2) HVSMOW-SLAP difference was 10-11 ‰ for modern collagen and 12-14 ‰ for hair, demonstrating that reactor configuration is important for these proteins, and that the use of a chromium-packed reactor may be desirable. In contrast, Bombyx mori cocoon (silk) δ(2) HVSMOW-SLAP values did not differ with reactor type. In general, δ(2) HVSMOW-SLAP and mass fraction H differences by reactor packing increased with mass fraction nitrogen in the sample. With the Cr-packed reactor hydrogen mass fractions were at theoretically expected values, while the glassy carbon reactor produced lower yields of hydrogen. CONCLUSIONS: The protein and amino acid δ(2) HVSMOW-SLAP values measured by factory-recommended online HTC methods differ from those from Cr-containing reactor packing. The magnitude of the differences is variable with sample type; the molecular structure and diagenetic history of each sample may be important. Careful attention to this effect is therefore recommended for the δ(2) H measurement for all nitrogen-containing analytes. Copyright © 2016 John Wiley & Sons, Ltd.

Limits and possibilities in the geolocation of humans using multiple isotope ratios (H, O, N, C) of hair from east coast cities of the USA.

We examined multiple natural abundance isotope ratios of human hair to assess biological variability within and between geographic locations and, further, to determine how well these isotope values predict location of origin. Sampling locations feature differing seasonality and mobile populations as a robust test of the method. Serially-sampled hair from Cambridge, MA, USA, shows lower δ(2)H and δ(18)O variability over a one-year time course than model-predicted precipitation isotope ratios, but exhibits considerable differences between individuals. Along a ∼13° north-south transect in the eastern USA (Brookline, MA, 42.3 ° N, College Park, MD, 39.0 ° N, and Gainesville, FL, 29.7 ° N) δ(18)O in human hair shows relatively greater differences and tracks changes in drinking water isotope ratios more sensitively than δ(2)H. Determining the domicile of humans using isotope ratios of hair can be confounded by differing variability in hair δ(18)O and δ(2)H between locations, differential incorporation of H and O into this protein and, in some cases, by tap water δ(18)O and δ(2)H that differ significantly from predicted precipitation values. With these caveats, randomly chosen people in Florida are separated from those in the two more northerly sites on the basis of the natural abundance isotopes of carbon, nitrogen, hydrogen, and oxygen.

Microwave Spectrum, Structure, and Hyperfine Constants of Kr-AgCl: Formation of a Weak Kr-Ag Covalent Bond.

The pure rotational spectrum of the complex Kr-AgCl has been measured between 8-15 GHz using a cavity pulsed-jet Fourier transform microwave spectrometer. The complex was found to be linear and relatively rigid, with a Kr-Ag bond length of approximately 2.641 Å. The Kr-Ag stretching frequency was estimated to be 117 cm(-1). Ab initio calculations performed at the MP2 level of theory gave the geometry, vibration frequencies, Kr-Ag bond dissociation energy, and orbital populations. The Kr-Ag bond dissociation energy was estimated to be approximately 28 kJ mol(-1). The Kr-Ag force constant and dissociation energy are greater than those of Ar-Ag in Ar-AgCl. The chlorine nuclear quadrupole coupling constants show slight changes on complex formation. Ab initio orbital population analysis shows a small shift in sigma-electron density from Kr to Ag on complex formation. The combined experimental and ab initio results are consistent with the presence of a weak Kr-Ag covalent bond. Copyright 2001 Academic Press.