Thomas C. Hoering

Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms

Two classes of procaryotic organisms were cultured on specific inorganic and organic nitrogenous substrates. The organisms fractionated these substrates in characteristic ways during the synthesis of their cellular biochemical compounds. Blue-green algae, Anabaena sp., were raised on molecular nitrogen, nitrate and ammonia in the presence of excess carbon dioxide. The difference between δ15N of either nitrate or ammonium and the δ15N of the algae grown on either source was 13%o. The δ15N of Anabaena that fixed N2 was 2% lighter than the nitrogen gas supplied to the algae. These fractionations are associated with enzymatic incorporation of the nitrogen into the cell. A heterotrophic bacterium, Vibrio harveyi was grown on a single amino acid as its source of both nitrogen and carbon. Cells grown on glutamic acid were enriched in 15N relative to substrate, whereas those grown on alanine were depleted in 15N compared to source nitrogen. The bacterial cultures were enriched in 13C relative to the substrate. These cultures were then hydrolyzed and individual amino acids isolated and isotopically analyzed. The isotopic compositions of the amino acids have a wide range of values; most appear to have isotope fractionations associated with the metabolic pathways in their synthesis. These results and the application of the coupled separation-isotopic analysis of amino acids yield a better understanding of comparative biochemistry for these organisms. Such analyses offer valuable information for the tracing of biosynthesis and early diagenesis to help explain the fossil record.

The isotopic composition of carbon and nitrogen in individual amino acids isolated from modern and fossil proteins

Pigs were reared in laboratory pens on controlled diets that consisted of either 100% C3 plants or 100% C4 plants. Carbon and nitrogen isotopic compositions of the diets, and the resulting pig products, purified collagen and muscle tissue, were measured to determine isotopic fractionation during growth and metabolism. Total collagen from pigs grown on C3 diets was enriched in 13C by 3·2‰ and in 15N by 2·2‰, whereas that from pigs reared on C4 diets was enriched in 13C by 1·4‰ and in 15N by 2·3‰. In addition, fractionation between pigs and their diets was determined at the molecular level on individual amino acids separated by ion exchange chromatography. The carbon isotopic compositions of separated amino acids from the C3 and C4 diets were transferred to amino acids in bone collagen. For nitrogen, the isotopic compositions of all nonessential amino acids were enriched in 15N relative to those amino acids in the diet. Threonine, an essential amino acid, behaved oppositely, in that its isotope ratio (δ15N) was depleted by an average of 6‰ from the δ15N of the whole collagen. Similar isotopic patterns were analysed in collagenous amino acids extracted from field specimens that included both herbivores and carnivores; marine animals and terrestrial animals; and C3 and C4 feeders. Amino acids from two fossil bones, a bison (4500 years old) and a whale (70,000 years old), recorded the same isotopic signals as modern collagen. The ubiquity of these isotopic patterns at the molecular level suggests that distinct biochemical mechanisms control the metabolism of amino acids in animals rather than random synthesis.

Differential fractionation of oxygen isotopes by cyanide-resistant and cyanide-sensitive respiration in plants

Stable-isotope discrimination factors (D) for the uptake of oxygen during respiration by a variety of plant materials were determined by measuring 18O enrichment in a closed system. Bakers yeast (Saccharomyces cerevisiae Meyer) and mitochondrial preparations from bakers yeast and from castor bean (Ricinus communis L.) endosperm, all of which are fully sensitive to cyanide, discriminated againt 18O by about 16–18‰. Whole Medicago sativa L. seedlings, isolated intact Asparagus sprengeri Regel mesophyll cells, and spadix mitochondria of Eastern skunk cabbage (Symplocarpus foetidus L.) had higher Ds of about 20–22‰. These materials all had some capacity for the cyanide-resistant alternative respiration pathway and in the presence of cyanide discriminated by about 24–26‰. When treated with salicylhydroxamic acid or tetraethylthiuram disulfide, which inhibit the alternative pathway, discrimination was about 17–19‰. Where respiration was limited by oxygen diffusion (slices of thermogenic tissues from S. foetidus and Sauromatum gutfatum Schott), fractionation was much reduced and the difference between the two respiratory pathways was masked. Isotope discrimination by soybean lipoxygenase (EC 1.13.11.12) supplied with linoleic acid was much lower than by respiration. Where diffusion is not a problem, the D value obtained in the absence of inhibitor can be used to estimate the partitioning of electron transport between the two pathways at steady-state by linear interpolation between the Ds characteristic of cyanide-resistant and cyanide-sensitive respiration.

Biogeochemistry of the stable hydrogen isotopes

Abstract The fractionation of H isotopes between the water in the growth medium and the organically bonded H from microalgae cultured under conditions, where light intensity and wavelength, temperature, nutrient availability, and the H isotope ratio of the water were controlled, is reproducible and light dependant. All studies were based either on the H isotope ratios of the total organic H or on the lipids, where most of the H is firmly bonded to C. H bonded into other macromolecules, proteins, carbohydrates and nucleic acids, does not exchange with water, when algae are incubated in water enriched with deuterium. Only after the destruction of quaternary H bonds are labile hydrogens in macromolecules free to exchange with water. By growing algae (18 strains), including blue-green algae, green algae and diatoms, in continuous light, the isotope fractionations in photosynthesis were reproducibly −93 to −178 %. depending on the organism tested. This fractionation was not temperature dependent. Microalgae grown in total darkness with an organic substrate did not show the isotope fractionation seen in cells grown in light. In both light- and dark-grown algae, however, additional depletion of deuterium (−30 to −60%.) in cellular organic matter occurs during the metabolism of carbohydrates to form lipids. Plants from several natural populations also fractionated isotopes during photosynthesis by an average of −90 to −110%. In addition, the organically bonded H in nonsaponifiable lipids was further fractionated by −80%. from that in saponifiable lipids, isolated from two geographically distinct populations of marsh plants. This difference between H isotope ratios of these two groups of lipids provides an endogenous isotopic marker.

The compositional limits of fluid immiscibility in the system H2ONaClCO2 as determined with the use of synthetic fluid inclusions in conjunction with mass spectrometry

Abstract The compositional limits of fluid immiscibility in the system NaClH2OCO2 were investigated from 500° to 700°C at pressures of 1, 2 and 3 kbar. Synthetic fluid inclusions formed in quartz prisms were equilibrated with high-temperature, high-pressure fluids in hydrothermal pressure vessels. The inclusions were analyzed optically, by mass spectrometry, and by microthermometry. The fields of immiscibility were defined as a function of temperature and pressure. Mass spectrometry was used to validate the existence of the immiscible fluids and to determine the CO 2 H 2 O ratios of the two coexisting phases. Microthermometry, i.e. the measurement of the melting temperature of solid sodium chloride, was used in conjunction with the results of the mass spectrometry measurements to define the tie-lines within the two-phase fields. Isochores and the location of a portion of the isopleth were determined for composition NaCl8.9H2O76.1CO215.0.

Carbon isotope geochemistry of graphite vein deposits from New Hampshire, U.S.A.

Abstract Graphite veins of hydrothermal origin occur throughout central New Hampshire. Veins truncate sillimanite-grade, metasedimentary rocks of Early Devonian-Silurian age and range in size from microscopic to meters in thickness. In addition to graphite, veins may contain quartz, tourmaline, ilmenite, rutile, sillimanite, muscovite or chlorite. Vein mineralogy is generally compatible with wall rock mineral assemblages. Mineralization structures include wall-rock alteration zones, coxcomb graphite crystals on vein walls, and botryoidal, concentrically layered graphite-silicate nodules. The δ13C values of graphite in 14 deposits studied range from − 28%. (PDB) to − 9%. Veins whose textures give evidence of a single stage of mineralization have a narrow range of δ13C values (± 0.2%.). Other veins record successive episodes of graphite precipitation and have ranges of 3–6%. In one sample, adjacent layers of graphite differ by 3%. The wide range of δ13C may be explained by mixing carbon from two crustal reservoirs: biogenic, reduced carbon and carbonate. Precipitation of graphite results from mixing two or more aqueous fluids with different CO 2 CH 4 ratios. Parental fluids are produced by devolatilization during metamorphism. Water-rich fluids with CH4 >CO2 and low δ13C are derived from pelites that contained organic matter; whereas fluids with CO2 >CH4 and high δ13C come from siliceous carbonates.

Composition of estuarine colloidal material: organic components

Abstract Colloidal material in the size range 1.2 nm to 0.4 μm was isolated by ultrafiltration from Chesapeake Bay and Patuxent River waters (U.S.A.). Temperature controlled, stepwise pyrolysis of the freeze-dried material, followed by gas chromatographic-mass spectrometric analyses of the volatile products indicates that the primary organic components of this polymer are carbohydrates and peptides. The major pyrolysis products at the 450°C step are acetic acid, furaldehydes, furoic acid, furanmethanol, diones and lactones characteristic of carbohydrate thermal decomposition. Pyrroles, pyridines, amides and indole (protein derivatives) become more prevalent and dominate the product yield at the 600°C pyrolysis step. Olefins and saturated hydrocarbons, originating from fatty acids, are present only in minor amounts. These results are consistent with the composition of Chesapeake phytoplankton (approximately 50% protein, 30% carbohydrate, 10% lipid and 10% nucleotides by dry weight). The pyrolysis of a cultured phytoplankton and natural particulate samples produced similar oxygen and nitrogencontaining compounds, although the proportions of some components differ relative to the colloidal fraction. There were no lignin derivatives indicative of terrestrial plant detritus in any of these samples. The data suggest that aquatic microorganisms, rather than terrestrial plants, are the dominant source of colloidal organic material in these river and estuarine surface waters.

The solubility of carbon monoxide in silicate melts at high pressures and its effect on silicate phase relations

Abstract The solubility of CO-CO 2 vapors in equilibrium with graphite has been measured in several silicate melts at pressures to 30 kbar and temperatures to 1700°C. The vapors contain 30–40% CO, by thermodynamic calculation. The CO-CO 2 vapors are slightly more soluble than CO 2 alone in melts investigated at near-liquidus temperatures. The temperature dependence of the solubility of CO is apparently negative, in contrast to the positive temperature dependence of CO 2 . As a result, the solubility of CO-CO 2 at superliquidus temperatures is less than that of CO 2 . Infrared spectra of quenched glasses containing CO and CO 2 essentially show only a CO 3 2− absorption band. The depressions of the liquidi of two compositions by CO-CO 2 vapor were found to be slightly greater than the depressions by CO 2 vapor. These phase relations imply that melting points are depressed more by CO than by CO 2 and that CO/(CO + CO 2 ) is somewhat greater in the liquid than in the vapor. The temperatures and pressures at which the liquidus phase of a peridotitic composition changes from olivine to orthopyroxene have been determined. These determinations suggest that melts containing CO and CO 2 are nearly as polymerized as melts containing only CO 2 . These melts are significantly more polymerized than volatile-absent melts. Melting relations of peridotite in the presence of CO-CO 2 will be similar, accordingly, to melting in the presence of CO 2 .

Isotope Fractionation during Oxygen Production and Consumption by Plants

Knowledge of isotope discrimination factors related to oxygen production and consumption by plants is important to an improved understanding of the global oxygen cycle. This information is also needed for the application of stable oxygen isotope methods, at natural abundance levels, to plant physiology and ecology.

Olefinic hydrocarbons from Bradford, Pennsylvania, crude oil

A distillation fraction of 2% of a crude oil from the Bradford, Pennsylvania, field was isolated as olefinic hydrocarbons. The olefins are approximately 40% normal, 30% branched chain, and 30% monocyclic. The double bonds are mainly in the trans configuration and are located in internal positions of long carbon chains. The carbon skeletons of the olefins and the coexisting saturated hydrocarbons correspond closely and have identifical 13C contents. The observed molecular structures can be accounted for by a process of natural thermal cracking of saturated hydrocarbons followed by a rapid disappearance of cis and terminal olefins.