Isotopic Tracers in Biology. An Introduction to Tracer Methodology

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A brief review of the time line in tools and capabilities is then given.


Next, focusing on the major stable isotopes of carbon, nitrogen, oxygen, silicon, and sulfur C, N, O, Si, and S —but biased toward our experience with C and N—examples of applications and lessons learned from these applications are highlighted. The perspective we aim to convey is that the field has advanced from focusing primarily on tool development and testing, to increasing use of isotopic abundance and labeling to address novel, difficult, and inconvenient questions.

Kinetic isotope effects underlie many of the differences in stable isotope ratios that we observe among ecosystem components, and fractionation associated with specific biogeochemical reactions, different isotopes, and different physiological states of organisms are often distinct, allowing fundamental processes to be interpreted based on changes in stable isotope values. The nomenclature of stable isotopes is quite varied.

Some of this variability relates to specific applications, but this does create potential for confusion.

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The vocabulary associated with enrichment studies is also different from that of the natural abundance stable isotope literature. Thus, 1. Thus: 2. Many geochemists prefer the definition above, with the lighter isotope in the denominator, while many chemists and biologists adopt the inverse formulation Fry Thus: 4. This kinetic isotope effect, or isotope discrimination factor, can be thought of as the ratio of rates with which isotopes are converted from reactant to product.

For unidirectional reactions in a closed system, kinetic isotopic fractionation follows that of a Rayleigh distillation model reviewed by Sigman and Casciotti ; Sigman et al. In general, the lighter isotope is incorporated into product at a faster rate than the heavier isotope, causing the residual reactant pool to become progressively more enriched in the heavier isotope with time.

When all of the heavy and light isotopes in the reactant pool have been converted to product, there is no longer an observable isotope effect. However, even when kinetic fractionation effects are strong, resolving the different processes that contribute to these effects remains challenging. Applications involving added isotope to trace enrichment during a process generally involve a different set of terms and assumptions e.

Atom fraction is an absolute measure of the number of atoms of the isotope relative to atoms of the element, the mole fraction of heavy isotope relative to heavy plus light. The velocity of the rate of uptake or transformation of an isotopic enrichment into a product V , also termed specific uptake rate, with units of reciprocal time, i.

Tracer experiments are commonly used in determining the uptake rate of N by phytoplankton. Collos suggested a modification to Eq. Glibert et al. The magnitude of errors associated with failure to apply such modifications depends on the type of sample e.

Radioactive tracer

Kinetic isotope effects in understanding of enrichment studies are typically ignored, as the magnitude of tracer addition generally overwhelms such effects and it is thought that introduced isotopes do not modify metabolic pathways. However, Andriukonis and Gorokhova have shown that there are profound differences in the growth of algae when provided 15 N enriched media.

Such effects may need to be re considered when very high isotope enrichment levels are used, as in experiments involving DNA or proteome labeling. For example, for N uptake, 6. In nitrification studies where the added isotope is NH 4 , the velocity of the rate is calculated the same way, but the target pool is the concentration of NO 2 or NO 3. In sum, isotope notation, regardless of preferred terms, is the consideration of ratios: ratios of the less abundant to the more abundant isotope, ratios in samples relative to standards, and ratios of enrichment relative to total compound availability.

Many final calculated values are, in fact, ratios of ratios of ratios. Moreover, common notations and calculations are derived to amplify the often very small differences between samples and standards. The importance of recognizing sources of error and assumptions in isotopic approaches is a theme returned to repeatedly throughout this article. Mass spectrometers have been in use for about a century for isotope analysis, initially largely in laboratories of a few renowned physicists, but the sophistication of isotope applications is rapidly advancing, and tools are quickly evolving Fig.

Pioneering work by Craig revealed that C isotope differences among organisms are primarily generated during primary production. Through seminal work by Goering et al. By applying 15 N tracers, mostly in the form of 15 NO 3 and 15 NH 4 , the understanding of environmental regulation of N uptake by phytoplankton was made possible.

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Early natural abundance work showed that C isotopes are transferred to consumers without fractionation DeNiro and Epstein , while N isotope ratios of consumers become enriched by a few per mil during trophic transfers DeNiro and Epstein ; Minagawa and Wada The latter studies provided the basis and rationale for the use of N isotopes in trophic transfer studies.

Size fractionated material e.

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  6. These analyses generally required a relatively large sample size, precluding analysis of single microbes, protists, or individual nematodes or zooplankton. Based on work by Carman and Fry , modifications to conventional elemental analysis and mass spectrometry approaches were developed that allowed for both 13 C and 15 N analysis in very small samples.

    Techniques for isolation or extraction of dissolved nutrients, e. Isotope dilution approaches allow the simultaneous resolution of two opposing processes, such as N incorporation and N regeneration in a natural sample. An enrichment of isotope is made, and its incorporation is measured in biomass. However, over time, the atom fraction of the isotope in dissolved form i. This is due to the fact that those organisms carrying out regeneration, such as microzooplankton, will release NH 4 that is not isotopically labeled as the short time scale of the experiment precludes this.

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    Sigman et al. Mass spectrometers advanced and the coupling of gas chromatographs GC with mass spectrometers were especially important Fig.

    The development of the continuous flow interface between an elemental analyzer and isotope ratio mass spectrometer allowed for automation of sampling by the instrument no longer did individual samples have to be prepped independently for injection into the mass spectrometer. Sample throughput increased many fold and environmental and ecological applications became routine.

    With more rapid and automated analysis, ecological and biogeochemical studies could now be replicated, and environmental gradients could be better resolved. The coupling of gas chromatographs and mass spectrometers also facilitated the dual analysis of C and N on the same sample. More affordable mass spectrometers, which included quadrupole mass spectrometers, became available, mostly in the early s Fig.

    Quadrupole mass spectrometers led the way to applications of membrane inlet mass spectrometry e. A MIMS instrument coupled to a quadrupole mass spectrometer is often used, for example, in applications of the isotope pairing method for denitrification and N 2 fixation e.

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    Here, rather than bulk isotopic analysis of particulate or aqueous samples, various biomarkers, such as lipids, carbohydrates, or amino acids AAs , are extracted and their isotope composition compared Boschker and Middelburg Biomarkers are compounds that ideally are specific to a species, or more usually a group of organisms. The CSIA approach can be used at natural abundance as well as for tracer applications.

    When coupled with other advancing methods, such as those of molecular biology, this approach can be very powerful in the analysis of microbial communities, and also of the diet of animals e. In food web studies, the ability to resolve AAs aids dietary reconstruction in that the 13 C and 15 N enrichment of individual AAs between food source and consumer varies widely, but essential AAs have distinct isotope fingerprints which may be passed through food webs without modification, giving clues of source identity. In addition to food web studies, CSIA is finding increasing application in studies of compound biodegradation where contaminants are of concern.

    Flow cytometric sorting of phytoplankton or bacterioplankton cells followed by isotope ratio mass spectrometry has been done both for natural abundance studies e. Recently, van Roij et al. The relatively modest investment and robustness of this method will further stimulate the use of stable isotopes in experimental and field studies. When coupled with other highly sensitive tools such as molecular approaches, information on microbial associations and other difficult to resolve pathways is now possible.

    An additional method gaining rapid application within microbial ecology is that of stable isotope probing SIP, e. In these methods, an enrichment is made with an isotopic compound, e. Nucleic acids with different densities can thus be separated and their differential incorporation of the label can be measured. Various molecular analyses of these labeled nucleic acids can be undertaken in tandem, using fingerprinting, clone libraries, metagenomics, and so on.

    By linking function and taxonomic identity, the responses and interactions of microorganisms to ecological conditions can be resolved at very fine scales. Collectively, these tools are rapidly increasing sophistication in biogeochemical studies, source tracking, food web analysis, and paleoecological studies.

    As will be shown in the next section, the examples of applications are wide and stable isotopes have become a critical part of a larger toolkit by which ecological, oceanographic, limnological, and paleoecological questions are addressed. Examples that are highlighted here were selected to provide a breadth of approaches, ecosystems, questions addressed, or insights gained. These categories are for convenience only, as many approaches and insights span multiple disciplines.

    Stable isotope applications for the study of rate processes and biogeochemical cycling are so extensive that justice cannot be done to all the insights that have been so gained; only a few snapshots are captured here. Quite simply, isotopes—and especially isotope enrichment studies—provide the foundation for our understanding of the biogeochemical pathways of nutrient cycling and the physiological rate processes associated with primary production and nutrient assimilation.

    An enormous literature exists with respect to applications of stable isotopes in studies of oceanic N cycles see reviews by Sigman and Casciotti ; Sigman et al.

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    Much is known regarding the isotopic characteristics of the predominant sources and sinks of N, and processes such as N 2 fixation, nitrification, and denitrification. The fixation of N 2 , which has been extensively studied in the ocean with a particular focus on Trichodesmium , introduces bioavailable N at isotopic values near atmosphere; i.

    Thus, using a mass balance approach, Montoya et al. This study showed that the contribution of atmospheric N was larger than anticipated, and there was a major dependence of the planktonic food web on this N derived by fixation, revealed by the calculated contribution of N from diazotrophs to four different size classes of plankton.

    Denitrification, in contrast to N 2 fixation, is a process with strong isotopic discrimination. Denitrifiers, in consuming NO 3 in the water column, use the lighter form of N at a faster rate than the heaver form of N, leaving NO 3 in the dissolved pool that can be substantially enriched with 15 N. In the sediment, however, denitrifiers are more often limited by available NO 3 , and consume most all of it, and thus do not leave a residual enriched pool of NO 3 and isotopic discrimination is not resolvable Brandes and Devol ; Sigman et al.

    Although the processes of NO 3 assimilation and denitrification have been shown to have fairly strong isotopic effects using N isotope analysis, unraveling those associated with the process of nitrification has proven to be more challenging. Nitrification also processes N 2 O as an intermediate. By using multiple isotopes within the same compound, isotopologues, different processes could be revealed. Moreover, dual isotope measurements of both NO 2 and NO 3 have revealed NO 3 regeneration in the euphotic zone and in O 2 deficient zones e.