Marilyn L. Fogel

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.

Isotope Fractionation during Primary Production

The biological source of sedimentary organic matter can be inferred from detailed chemical studies on the structure of individual molecules extracted from sediments. These inferences are drawn from established relationships between biological source materials and diagenetically altered compounds (de Leeuw and Largeau, this volume, Chapter 2). A second method for inferring the biological source and the ecological setting in which organisms existed is with isotopic tracers of carbon, nitrogen, oxygen, hydrogen, and sulfur in organic matter and inorganic substances that have been processed by living organisms. Stable isotope compositions of bulk organic matter have integrative signals from processes that have occurred over the life of the organism, whereas those of individual compounds can record specific events in life. In this chapter, we will review recent advances in biological isotope fractionations of the light stable isotopes and attempt to offer an interpretation of processes that affected the isotopic signature of organic substances in living organisms that were preserved in 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.

Photosynthetic Fractionation of the Stable Isotopes of Oxygen and Carbon

Isotope discrimination during photosynthetic exchange of O2 and CO2 was measured using enzyme, thylakoid, and whole cell preparations. Evolved oxygen from isolated spinach thylakoids was isotopically identical (within analytical error) to its source water. Similar results were obtained with Anacystis nidulans Richter and Phaeodactylum tricornutum Bohlin cultures purged with helium. For consumptive reactions, discrimination ([delta], where 1 + [delta]/1000 equals the isotope effect, k16/k18 or k12/k13) was determined by analysis of residual substrate (O2 or CO2). The [delta] for the Mehler reaction, mediated by ferredoxin or methylviologen, was 15.3[per mille (thousand) sign]. Oxygen isotope discrimination during oxygenation of ribulose-1,5-bisphosphate (RuBP) catalyzed by RuBP carboxylase/oxygenase (Rubisco) was 21.3[per mille (thousand) sign] and independent of enzyme source, unlike carbon isotope discrimination: 30.3[per mille (thousand) sign] for spinach enzyme and 19.6 to 23[per mille (thousand) sign] for Rhodospirillum rubrum and A. nidulans enzymes, depending on reaction conditions. The [delta] for O2 consumption catalyzed by glycolate oxidase was 22.7[per mille (thousand) sign]. The expected overall [delta] for photorespiration is about 21.7[per mille (thousand) sign]. Consistent with this, when Asparagus sprengeri Regel mesophyll cells approached the compensation point within a sealed vessel, the [delta]18O of dissolved O2 came to a steady-state value of about 21.5[per mille (thousand) sign] relative to the source water. The results provide improved estimates of discrimination factors in several reactions prominent in the global O cycle and indicate that photorespiration plays a significant part in determining the isotopic composition of atmospheric oxygen.

Nitrogen-isotope compositions of metasedimentary rocks in the Catalina Schist, California: Implications for metamorphic devolatilization history

Abstract In the Catalina Schist subduction-zone metamorphic complex (California), metasedimentary rocks show a decrease in N concentration and an increase in δ15Nair with increasing metamorphic grade. Lowest-grade lawsonite-albite rocks contain 632 ± 185 ppm N with δ 15 N = +1.9 ± 0.6‰ , whereas highgrade amphibolite equivalents contain 138 ± 76 ppm N with δ 15 N = +4.3 ± 0.8‰ . Loss of N accompanied devolatilization reactions that evolved H2O-rich C-O-H-S-N fluids through consumption of chlorite and phengitic white mica and production of mineral assemblages containing muscovite, biotite, garnet and kyanite. Whole-rock N concentrations of up to 200 ppm in veins and pegmatites produced during high- P T metamorphism reflect the redistribution of N during devolatilization and partial melting of the metasedimentary rocks. Bulk fluid-rock N-isotope fractionations (Δ15N = δ15Nfluid − δ15Nrock) of −1.5 ± 1‰ were calculated with the Rayleigh distillation equation, taking into account variability in rock composition by comparison of samples with similar K2O concentrations. These fractionations are similar to but slightly lower than published calculated fractionations for N2-NH4+ exchange at the temperature range of 350–600°C over which most of the devolatilization occurred in the Catalina Schist (approximately −3.4 to −2.25‰). The N systematics appear to be explained by N2-NH4+ exchange and a devolatilization process intermediate in behavior to batch volatilization and Rayleigh distillation. The observed shifts in N concentration and δ15N cannot be explained by NH3-NH4+ exchange at these temperatures using the equilibrium models. The distillation devolatilization process implicated in this study may govern the behavior of other trace elements partitioned into hydrous fluids during devolatilization (e.g., B, Cs, U). Similarity of the calculated fluid δ15N( ~ −1.5 to +5.5‰) with compositions of natural gases inferred to be derived from metasedimentary sources indicates the possibility of using N as a tracer of large-scale volatile transport.

The Provenances of Asteroids, and Their Contributions to the Volatile Inventories of the Terrestrial Planets

Constraining the Birthplace of Asteroids Many primitive meteorites originating from the asteroid belt once contained abundant water that is now stored as OH in hydrated minerals. Alexander et al. (p. 721, published online 12 July) estimated the hydrogen isotopic compositions in 86 samples of primitive meteorites that fell in Antarctica and compared the results to those of comets and Saturns moon, Enceladus. Water in primitive meteorites was less deuteriumrich than that in comets and Enceladus, implying that, in contradiction to recent models of the dynamical evolution of the solar system, the parent bodies of primitive meteorites cannot have formed in the same region as comets. The results also suggest that comets were not the principal source of Earths water. Hydrogen isotopic analysis of primitive meteorites implicates asteroids as early sources of Earth’s water. Determining the source(s) of hydrogen, carbon, and nitrogen accreted by Earth is important for understanding the origins of water and life and for constraining dynamical processes that operated during planet formation. Chondritic meteorites are asteroidal fragments that retain records of the first few million years of solar system history. The deuterium/hydrogen (D/H) values of water in carbonaceous chondrites are distinct from those in comets and Saturn’s moon Enceladus, implying that they formed in a different region of the solar system, contrary to predictions of recent dynamical models. The D/H values of water in carbonaceous chondrites also argue against an influx of water ice from the outer solar system, which has been invoked to explain the nonsolar oxygen isotopic composition of the inner solar system. The bulk hydrogen and nitrogen isotopic compositions of CI chondrites suggest that they were the principal source of Earth’s volatiles.

Renewable and nonrenewable resources: Amino acid turnover and allocation to reproduction in Lepidoptera

The allocation of nutritional resources to reproduction in animals is a complex process of great evolutionary significance. We use compound-specific stable isotope analysis of carbon (GC/combustion/isotope ratio MS) to investigate the dietary sources of egg amino acids in a nectar-feeding hawkmoth. Previous work suggests that the nutrients used in egg manufacture fall into two classes: those that are increasingly synthesized from adult dietary sugar over a females lifetime (renewable resources), and those that remain exclusively larval in origin (nonrenewable resources). We predict that nonessential and essential amino acids correspond to these nutrient classes and test this prediction by analyzing egg amino acids from females fed isotopically distinct diets as larvae and as adults. The results demonstrate that essential egg amino acids originate entirely from the larval diet. In contrast, nonessential egg amino acids were increasingly synthesized from adult dietary sugars, following a turnover pattern across a females lifetime. This study demonstrates that female Lepidoptera can synthesize a large fraction of egg amino acids from nectar sugars, using endogenous sources of nitrogen. However, essential amino acids derive only from the larval diet, placing an upper limit on the use of adult dietary resources to enhance reproductive success.

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.

Variability in the preservation of the isotopic composition of collagen from fossil bone

Abstract Collagen from bone was prepared by several methods. For modern and well-preserved bone the δ13C and δ15N of collagen replicas obtained after HCl or EDTA demineralization were similar to those obtained with a gelatinization procedure. However, in more poorly preserved fossil bone the δ13C and δ15N varied among the different protein extracts. The yield of collagen obtained with EDTA demineralization was consistently higher than extraction procedures that used HCl. The δ13C of individual amino acids separated from the collagen of modern and fossil whale bone varied up to 17%., and the δ15N from the same amino acids ranged over 47%. The δ13C and δ15N of most amino acids clustered closely to the average of the HCl insoluble collagen. The δ13C of the major amino acid in collagen, glycine, differed from the average HCl insoluble collagen by approximately 8%. in the fossil whale and 14%. in the modern whale. The δ15N of glycine differed from the average HCl insoluble values by approximately 4%. in the fossil whale and 7%. in the modern whale. Thus, diagenetic changes that alter the ratio of glycine to other amino acids in bone can be expected to perturb the values for carbon and nitrogen isotopes.

Isotopic Tracking of Change in Diet and Habitat Use in African Elephants

The carbon, nitrogen, and strontium isotope compositions of elephants in Amboseli Park, Kenya, were measured to examine changes in diet and habitat use since the 1960s. Carbon isotope ratios, which reflect the photosynthetic pathway of food plants, record a shift in diet from trees and shrubs to grass. Strontium isotope ratios, which reflect the geologic age of bedrock, document the concentration of elephants within the park. The high isotopic variability produced by behavioral and ecological shifts, if it is representative of other East African elephant populations, may complicate the use of isotopes as indicators of the source region of ivory.