We compiled published data on hydrogen isotope values for leaf wax n-alkanes (C 27 , C 29 ,

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Materials and methods Sample preparation All outcrop samples were collected on a field trip to South China in March 2007. Large blocks (>200 g) of the freshest exposures were deliberately targeted for sampling. The large rock blocks were subsequently trimmed by a water-cooled rock saw or by hammer in the laboratory to remove the potentially weathered surfaces and then broken into small pieces. The pieces were further crushed into powders using a SPEX 8515 Shatterbox with an alumina (ceramic) puck. Rock pieces that contained readily visible pyrite nodules or bands were discarded prior to crushing. Total organic carbon (TOC) and total inorganic carbon (TIC) TOC was determined as the difference between total carbon (TC) and total inorganic carbon (TIC) measured using a CS-500 carbon/sulfur analyzer with a hightemperature furnace and acidification module (Eltra, Germany). For TC, ~100 mg of sample powder were weighed into a ceramic boat and combusted in pure (99.95%) O 2 at 1350 °C for ~3 min. The total carbon liberated was then measured by infrared spectral absorption of the evolved CO 2. For TIC, ~100 mg of sample powder were reacted with 20% HCl, heated at 50 °C and stirred. TIC was also quantified by infrared absorption detection of the CO 2 generated. Analytical errors for TOC and TIC are ±0.1 wt% based on analysis of carbonate standard AR1034 (Alpha, USA). Pyrite sulfur isotopes (δ 34 S py) and concentrations Disseminated pyrite concentrations and isotopic compositions were analyzed by the chromium reduction method (S1). Pyrite extraction was performed under N 2 by the addition of 20 ml of concentrated HCl and 40 ml of 1M chromous chloride solution. The reaction mixture was heated for 2 h, with the liberated sulfide collected either as silver sulfide after bubbling through 30 ml of 3 wt% silver nitrate solution with 10% NH 4 OH by volume (for isotopic analysis) or as zinc sulfide after bubbling through 30 ml of 3 wt% zinc acetate with 10% NH 4 OH by volume for pyrite-S concentration. Mean recovery of parallel replicate pure pyrite standards was 105.6%. Filtered, rinsed and dried Ag 2 S precipitates were combined with an excess of V 2 O 5 and analyzed for S-isotope composition following online combustion using a Thermo Instruments Delta V Plus isotope ratio mass spectrometer coupled with a Costech elemental analyzer at the University of California, Riverside. Sulfur isotope compositions are expressed as δ 34 S = (R sample /R standard-1) × 1000, where R is the ratio of 34 S/ 32 S, reported as permil (‰)

Large hydrogen-isotopic (D/H) fractionations between lipids and growth water have been observed in most organisms studied to date. These fractionations are generally attributed to isotope effects in the biosynthesis of lipids, and are frequently assumed to be approximately constant for the purpose of reconstructing climatic variables. Here, we report D/H fractionations between lipids and water in 4 cultured members of the phylum Proteobacteria, and show that they can vary by up to 500‰ in a single organism. The variation cannot be attributed to lipid biosynthesis as there is no significant change in these pathways between cultures, nor can it be attributed to changing substrate D/H ratios. More importantly, lipid/water D/H fractionations vary systematically with metabolism: chemoautotrophic growth (approximately ؊200 to ؊400‰), photoautotrophic growth (؊150 to ؊250‰), heterotrophic growth on sugars (0 to ؊150‰), and heterotrophic growth on TCA-cycle precursors and intermediates (؊50 to ؉200‰) all yield different fractionations. We hypothesize that the D/H ratios of lipids are controlled largely by those of NADPH used for biosynthesis, rather than by isotope effects within the lipid biosynthetic pathway itself. Our results suggest that different central metabolic pathways yield NADPH-and indirectly lipids-with characteristic isotopic compositions. If so, lipid ␦D values could become an important biogeochemical tool for linking lipids to energy metabolism, and would yield information that is highly complementary to that provided by 13 C about pathways of carbon fixation.fatty acids ͉ fractionation ͉ metabolism ͉ hydrogen isotopes T he hydrogen-isotopic composition ( 2 H/ 1 H or D/H ratio, commonly expressed as a ␦D value) of lipids is being explored by scientists with diverse interests, including the origins of natural products (1, 2), biogeochemical cycles (3), petroleum systems (4), and paleoclimate (5-7). Because the D/H ratios of lipids are generally conserved over Ϸ10 6 -year time scales (8), they are a potentially useful tracer of biogeochemical pathways and processes in the environment. Most research to date has focused on higher plants, in which environmental water is the sole source of external hydrogen and consequently provides primary control over the D/H ratio of biosynthesized lipids (9). Although ␦D values for plant lipids and environmental water are generally well correlated, they are also substantially offset from each other. The biochemical basis for this lipid/water fractionation is not well understood. It is generally assumed to arise from a combination of isotope effects during photosynthesis and the biosynthesis of lipids (9-12), and is often treated as approximately constant to reconstruct isotopic compositions of environmental water as a paleoclimate proxy.There is, however, mounting evidence that the net D/H fractionation between lipids and water can vary by up to 150‰ in plants, even in the same organism (12-16). Modest fractionations associated with fatty acid elongation and desaturation ...

Isotope-ratio detection for gas chromatographyInstrumentation and methods exist for highly precise analyses of the stable-isotopic composition of organic compounds separated by GC. The general approach combines a conventional GC, a chemical reaction interface, and a specialized isotoperatio mass spectrometer (IRMS). Most existing GC hardware and methods are amenable to isotope-ratio detection. The interface continuously and quantitatively converts all organic matter, including column bleed, to a common molecular form for isotopic measurement. C and N are analyzed as CO 2 and N 2 , respectively, derived from combustion of analytes. H and O are analyzed as H 2 and CO produced by pyrolysis/reduction. IRMS instruments are optimized to provide intense, highly stable ion beams, with extremely high precision realized via a system of differential measurements in which ion currents for all major isotopologs are simultaneously monitored. Calibration to an internationally recognized scale is achieved through comparison of closely spaced sample and standard peaks. Such systems are capable of measuring 13 C/ 12 C ratios with a precision approaching 0.1? (for values reported in the standard delta notation), four orders of magnitude better than that typically achieved by conventional "organic" mass spectrometers. Detection limits to achieve this level of precision are typically a1 nmol C (roughly 10 ng of a typical hydrocarbon) injected on-column. Achievable precision and detection limits are correspondingly higher for N, O, and H, in that order.