Influence of diet on the distribution of nitrogen isotopes in animals

The influence of diet on the distribution of nitrogen isotopes in animals was investigated by analyzing animals grown in the laboratory on diets of constant nitrogen isotopic composition. 
The isotopic composition of the nitrogen in an animal reflects the nitrogen isotopic composition of its diet. The δ^(15)N values of the whole bodies of animals are usually more positive than those of their diets. Different individuals of a species raised on the same diet can have significantly different δ^(15)N values. The variability of the relationship between the δ^(15)N values of animals and their diets is greater for different species raised on the same diet than for the same species raised on different diets. Different tissues of mice are also enriched in ^(15)N relative to the diet, with the difference between the δ^(15)N values of a tissue and the diet depending on both the kind of tissue and the diet involved. The δ^(15)N values of collagen and chitin, biochemical components that are often preserved in fossil animal remains, are also related to the δ^(15)N value of the diet. 
The dependence of the δ^(15)N values of whole animals and their tissues and biochemical components on the δ^(15)N value of diet indicates that the isotopic composition of animal nitrogen can be used to obtain information about an animal's diet if its potential food sources had different δ^(15)N values. The nitrogen isotopic method of dietary analysis probably can be used to estimate the relative use of legumes vs non-legumes or of aquatic vs terrestrial organisms as food sources for extant and fossil animals. However, the method probably will not be applicable in those modern ecosystems in which the use of chemical fertilizers has influenced the distribution of nitrogen isotopes in food sources. 
The isotopic method of dietary analysis was used to reconstruct changes in the diet of the human population that occupied the Tehuacan Valley of Mexico over a 7000 yr span. Variations in the δ^(15)C and δ^(15)N values of bone collagen suggest that C_4 and/or CAM plants (presumably mostly corn) and legumes (presumably mostly beans) were introduced into the diet much earlier than suggested by conventional archaeological analysis.

Influence of Diet On the Distribtion of Nitrogen Isotopes in Animals

Virtually all nonequilibrium electron transfers on Earth are driven by a set of nanobiological machines composed largely of multimeric protein complexes associated with a small number of prosthetic groups. These machines evolved exclusively in microbes early in our planet's history yet, despite their antiquity, are highly conserved. Hence, although there is enormous genetic diversity in nature, there remains a relatively stable set of core genes coding for the major redox reactions essential for life and biogeochemical cycles. These genes created and coevolved with biogeochemical cycles and were passed from microbe to microbe primarily by horizontal gene transfer. A major challenge in the coming decades is to understand how these machines evolved, how they work, and the processes that control their activity on both molecular and planetary scales.

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The Microbial Engines That Drive Earth's Biogeochemical Cycles

▪ Abstract The use of stable isotope techniques in plant ecological research has grown steadily during the past two decades. This trend will continue as investigators realize that stable isotopes can serve as valuable nonradioactive tracers and nondestructive integrators of how plants today and in the past have interacted with and responded to their abiotic and biotic environments. At the center of nearly all plant ecological research which has made use of stable isotope methods are the notions of interactions and the resources that mediate or influence them. Our review, therefore, highlights recent advances in plant ecology that have embraced these notions, particularly at different spatial and temporal scales. Specifically, we review how isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen our understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies. Where possible we also...

Stable Isotopes in Plant Ecology

Biological N2 fixation represents the major source of N input in agricultural soils including those in arid regions. The major N2-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N2-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N2-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research.

Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in an Arid Climate

The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

I. The Diazotrophs.- 1 Recognition.- I. Historical Roots.- A. The Agricultural Imperative.- B. Early Experimentation.- II. Detection of Diazotrophs and Nitrogenase.- A. 15N2 Method.- B. Ammonia and Manometric Methods.- C. Acetylene Reduction Method.- D. Other Methods.- 2 Description and Classification of Diazotrophs.- I. Free-Living Diazotrophs.- A. Taxonomy and Characteristics.- 1. Bacteria.- 2. Blue-Green Algae.- B. Associative Symbioses.- 1. Loose Associations.- a) Interactions Among Microorganisms.- b) Phylloplane.- c) Rhizosphere.- 2. Associations Involving Morphological Modification or Accommodation.- 3. Associations with Animals.- II. Obligatory Symbioses.- A. Non-Legume Angiosperms.- B. Legumes.- 1. Rhizobia.- 2. Establishment of an Effective Legume Symbiosis.- 3. Tissue Culture Symbiosis.- Concluding Remarks.- 3 Perspectives.- I. Evolution.- A. N2 Fixation in Free-Living Organisms.- B. N2 Fixation in Symbioses.- II. Role of Diazotrophs in Global Nitrogen Economy.- A. Nitrogen Cycle.- 1. Nitrogen Sinks.- 2. Nitrogen Transformations and Transfers.- a) Soil-Water Pool.- b) Atmosphere.- Concluding Remarks.- B. Amount of N2 Fixed.- 1. N2 Fixation Rates in Various Ecosystems.- a) Cultivated Land.- b) Forests and Grasslands.- c) Unused Land.- d) Marine.- 2. Comparison with Previous Estimates.- II. Biochemistry and Biophysics.- 4 An Equation for the Nitrogenase Reaction.- I. The Clostridium - Pyruvate N2-Fixing System.- II. ATP.- III. Azotobacter Nitrogenase and Dithionite.- IV. H2 Evolution and ATP Hydrolysis: Nitrogenase Activity without N2.- V. Alternate Substrates for Nitrogenase.- VI. H2, N2, Hydrogenase and Nitrogenase - A Special Relationship?.- 5 Nitrogenase: The Catalyst.- I. Procurement.- A. Factors Affecting Nitrogenase Biosynthesis.- 1. Genetic Basis.- 2. Culture Conditions.- a) Metabolic Control.- b) General Considerations.- B. Extraction and Purification of Nitrogenase.- II. Properties of Mo-Fe Protein and Fe Protein.- A. Nitrogenase Proteins of Various Diazotrophs.- 1. Azotobacter vinelandii.- a) Mo-Fe Protein.- b) Fe Protein.- 2. Clostridium pasteurianum.- a) Mo-Fe Protein.- b) Fe Protein.- 3. Kiebsiella pneumoniae.- a) Mo-Fe Protein.- b) Fe Protein.- 4. Azotobacter chroococcum.- 5. Mycobacterium flavum.- 6. Chloropseudomonas ethylicum.- 7. Soybean Bacteroids.- B. Summary of Characteristics of the Nitrogenase Proteins.- 1. Mo-Fe Protein.- 2. Fe Protein.- III. Properties of Nitrogenase.- A. The Question of Nitrogenase Composition.- B. Physical-Chemical Characteristics of Nitrogenase.- C. Stability of Nitrogenase.- 1. O2 Sensitivity.- 2. Cold Lability.- D. Integrity of Reconstituted Nitrogenases.- 6 Nitrogenase: The Catalysis.- I. Nitrogenase Turnover.- A. Enzyme Concentration and Composition.- B. ATP.- 1. ATP and Reaction Rate.- a) Relationship of ATP, Enzyme Concentration and Activity.- b) Michaelis Constant.- c) ADP and Salt Inhibition.- 2. The ATP:2-Electron Ratio.- 3. Reductant-Independent ATP Hydrolysis.- 4. Other ATP Effects.- C. Dithionite.- D. External Factors.- 1. pH.- 2. Temperature.- 3. Inhibitors.- II. Substrate Reduction.- A. Electron Allocation: H3O+vs. Exogenous Reducible Substrate.- B. Specificity and Inhibition.- C. The Reducible Substrates.- 1. N2.- 2. Azide.- 3. N2O.- 4. Nitriles.- a) Cyanide.- b) Alkyl Cyanides.- c) Alkenyl Cyanides.- 5. Isonitriles.- 6. Alkynes.- Concluding Remarks on Substrate Reduction.- 7 Mechanism.- I. A Model for the Formation of Catalytically Active Species.- A. Protein and Ligand Complexes of the Model.- 1. Fe Protein * ATP * Mg Complex.- 2. Mo-Fe * Protein * Fe Protein Complexes.- B. Reactivity of the Model.- 1. Dilution Effect.- a) The Need for 2 Fe Proteins.- b) Effect of ATP Concentration.- c) ADP and Salt Effects.- 2. ATP Hydrolysis.- 3. Nature of ATP Saturation Curves and Reciprocal Plots for Km (ATP) Evaluation.- 4. Reaction of ATP with the Mo-Fe Protein * Fe Protein Complexes.- C. Allosteric Implications.- D. The Mechanistic Enigmas of ATP Hydrolysis and Electron Transfer.- II. Reducible Substrate Complexation and Reduction.- A. Locus of Active Site.- B. Role of Metals and Implications of Abiological Systems.- 1. Nitriding Systems.- 2. N2 Complexes in Aprotic Media.- 3. Homogeneous Protonic Catalysis.- C. Active Site Model and Reduction Sequence.- 8 Cellular Accommodation of Nitrogenase.- I. Localization of Nitrogenase.- II. Electron-Donating Systems.- A. Clostridium pasteurianum.- B. Azotobacter vinelandii.- C. Azotobacter chroococcum.- D. Mycobacterium flavum.- E. Bacillus polymyxa.- F. Chromatium.- G. Chloropseudomonas ethylicum.- H. Anabaena cylindrica.- I. Soybean Nodule Bacteroids.- III. Ammonia Assimilation.- IV. Other Enzymes and Proteins Associated with N2 Fixation.- A. Hydrogenase.- B. Leghemoglobin.- C. Other Proteins.- 1. Cytochromes.- 2. Nitrate Reductase.- References.

Nitrogen Fixation in Bacteria and Higher Plants

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of NO3−, K+, and H2PO4− into 3- to 4-day- and 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.

Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense.

The various nitrogenous solutes important to embryo development in symbiotic soybean plants were determined during the midpodfilling stage. Glutamine was the principal form of nitrogen, contributing 55% of the embryo nitrogen requirement. Asparagine was the second most important, contributing 20%. The ureides allantoin and allantoic acid directly contributed only insignificantly to the total nitrogen requirement of the embryo. These conclusions were based upon analyses of tissue extracts, translocation studies of radiolabeled solutes, analysis of in vivo seed coat exudate collected from the freespace of attached, surgically altered seeds, and the in vitro culture of isolated immature soybean embryos.

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Ralph W. F. Hardy

Papers

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Nitrogen Fixation in Bacteria and Higher Plants

Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense.

Role of Amides, Amino Acids, and Ureides in the Nutrition of Developing Soybean Seeds