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Stable isotope analysis of light elements, namely those of carbon, nitrogen, oxygen, sulphur and hydrogen, are increasingly used in research across a wide range of topics including soil science. Stable isotope analysis offers a unique tool to study biochemical processes occurring in soil ecosystems.
Principle of stable isotope analysis
An isotope is an atom whose nuclei contain the same number of protons but a different number of neutrons. Isotopes are broken into two specific types: stable and unstable. Unstable isotopes are commonly referred to as radioactive isotopes. There are approximately 300 known naturally occurring stable isotopes. Most of the light elements contain different proportions of at least two isotopes. Usually one isotope is the predominantly abundant isotope.
Traditionally, stable isotope analysis occupies itself with the so-called light stable isotopes, which are the basic elements of most biochemical processes. In figure 1, the average abundance of the naturally occurring light stable isotopes is shown.
Since most isotopic measurements are differential measurements and because the interesting isotopic differences between natural samples usually occur at and beyond the third significant figure of the isotope ratio, it has become conventional to express isotopic abundances using a differential notation. To provide a concrete example, it is far easier to say – and remember – that the isotope ratios of samples A and B differ by one part per thousand than to say that sample A has 0.3663 %15N and sample B has 0.3659 %15N. The notation that provides this advantage is indicated in general form below. δAXstd = 1000 x ((ARsample-ARstd)/ARstd)
This equation defines an explicit relationship between the abundance of isotope A of element X in a given sample and its abundance in a particular standard (designated by a subscript, here shown in general form as std). Values of δAXstd are usually expressed in parts per thousand. The corresponding symbol, ‰, is called ‘permil’.
The variations in the naturally occurring isotope ratios of compounds are small, but indicative of the processes through which they were formed. Heavy isotopes undergo all of the same chemical reactions as light isotopes, but simply because they are heavier reactions rates are slightly different. The tiny differences in reaction rate are called isotopic fractionation and cause the products of reactions to have different isotope ratios than the source materials.
Fractionation is caused by the differences in the chemical and physical properties of a certain atomic mass and concerns the concepts of isotope exchange and kinetic processes in reaction rates. Changes in temperature are just an example of an isotope exchange process that can cause isotopic fractionation.
Measuring stable isotopes
Differential measurement of isotopic abundances requires that each sample be compared to a standard e.g. a reference point which differs from the sample, if at all, only in its isotopic composition. For example, precise comparisons of isotopic compositions of carbonates can be made by comparing mass spectra of samples of carbon dioxide prepared from each material. The mechanism of comparison is neither so obvious nor so simple when isotopic compositions of organic materials are of interest, though there is nothing fundamentally wrong with comparing isotopic compositions of organic materials directly.
For example, the difference in carbon-isotopic compositions of two samples of benzene (C6H6, molecular weight = 78) might be measured by comparison of the (mass 79)/(mass 78) ion-current ratios, and if both samples were not available at the same time, each might be satisfactorily compared to an intermediate standard. The impracticality of this simple approach becomes clear, however, when it is recognised that:
• The measurement would be invalid if the hydrogen-isotopic compositions of the samples differed significantly
• An isotopic standard would be required for each compound of interest if indirect comparisons were to be made
• Comparisons between different compounds would be difficult and subject to systematic errors, and
• It would be impossible to make isotopic measurements on species that could not be readily volatilized to yield ion beams that were both intense and stable
Many advantages are gained by converting organic materials to ‘common denominator’ forms especially suitable for mass spectrometry and isotopic analysis. For example, the gases H2, N2, and O2 are ideal for hydrogen, nitrogen, and oxygen isotopic analyses because they are volatile and contain information only about isotope ratios for that element. There is, unfortunately, no analogous form of carbon. Carbon tetrafluoride, CF4, would be a logical choice because fluorine has no isotopes and quantitative details of the mass spectrum therefore depend only on carbon isotopic abundances. Carbon dioxide is easier to prepare, but contains exchangeable atoms of an element other than carbon.
The isotopic composition of the substance employed in the differential measurement (e.g. the compound used in isotope-ratio mass spectrometry) must faithfully represent that of the parent material. Fulfilment of this requirement for isotopic fidelity is assured if the entire material is quantitatively converted to the form used for analysis, but quantitative yields in preparative organic reactions are rare.
Fortunately, combustion can easily be driven to completion. It may be unusual to think of combustion as a preparative reaction, but it offers a remarkable combination of fundamental appeal (ease of obtaining quantitative yield) and experimental convenience. Carbon dioxide may be theoretically inferior to CF4 as a material for isotopic mass spectrometry, but it is far easier to prepare in high yield. The following table gives an overview of the form under which most elements are measured.
For the measurement of stable isotopes a special form of mass spectrometer is required, namely the isotope ratio mass spectrometer (IRMS). The basic concepts that govern isotope ratio mass spectrometry are the same that govern analytical mass spectrometry.
A gas source mass spectrometer comprises a source, flight tube and collectors (Figure 2). To analyse a sample gas, the molecules must be ionised in the source, and the ions are then formed into a beam and accelerated by an electric field. The ions then pass from the source into the flight tube, where they are magnetically deflected, and finally they are detected by the collector.
The ionisation is commonly achieved by passing a beam of electrons through the gas sample. Collision between, or close approach of an electron and a sample molecule can cause one or more electrons either to adhere to the molecule and form a negative ion, or to detach from the molecule and generate a positive ion. Isotope analysis usually involves the singly-charged positive ions (molecules that have lost one electron). The formation process is called electron impact.
The radical cation products are directed towards the mass analyser by a repeller electrode. The ions are accelerated and formed into a well-defined beam by raising the ionisation chamber to a positive potential and accelerating the ions out through a defining slit called the source slit. The ions are then passed towards a second defining slit at ground potential called the alpha slit, which eliminates unwanted ions, and enters the flight tube.
The flight tube forms the arc of a circle that passes between the poles of the magnet. As the ions travel down the tube, they are separated into beams of different radii corresponding to different masses. For singly-charged ions, the radius is determined by the nature of the magnetic and electric field. The combination of fields selects ions of particular mass and forms a mass filter. This principle is the basis of all magnetic-sector mass spectrometers. The ion beam to be measured passes through a slit called the resolving slit into the collector. A particular radius and hence mass is selected by the combination of the alpha slit at the source end of the flight tube and the resolving slit at the collector end.
In the collector, ions of the chosen mass are transmitted through the resolving slit and detected by a Faraday cup. The ion current from the cup is proportional to the number of incident ions and hence to the partial pressure of the corresponding isotopic molecular species in the sample gas. Multiple
Faraday cups are frequently used to obtain simultaneous detection of different isotopes. The most pronounced difference between analytical and isotope ratio instruments lies in the peak shape observed by scanning the magnetic or electric fields.
An analytical instrument provides a spectrum of mass peaks that is characteristic of chemical composition. Very narrow peaks are used to distinguish closely spaced masses. In isotope ratio work, the chemical composition of the sample is known and the fields are held constant. The variation of isotopes in an element is most precisely measured by using broad peaks, as these provide the most stable amplitude measurements.
The distinguishing characteristics of modern isotope-ratio mass spectrometers are (i) very high efficiency of ionisation and very intense ion beams (ii) multiple-collector systems allowing simultaneous collection of two or more ion beams, and (iii) dual – or even triple – inlet systems designed to allow rapid exchange of one sample gas for another in the ion source of the instrument. All of these features, unique to isotope-ratio instruments, are of special importance.
Stable isotopes in soil science
Soil is an integral part of terrestrial ecosystems. Many soil ecologists interested in soil ecosystem functioning rely, to some degree, on stable isotope methodologies. Significant advances have been made in the development and application of isotopic techniques in soil science studies. The measurement of natural variations in the abundance of stable isotopes of hydrogen, carbon, nitrogen and sulphur in soil, water and plant components can help to identify the source of water and nutrients used by plants and to quantify water and nutrient fluxes through and beyond the plant rooting zone as influenced by different irrigation and land management practices. These developments have been possible due to the increased sensitivity of continuous flow isotope-ratio mass spectrometers (IRMS) for analysing the isotopic composition in soil–plant–water components.
Hydrology
The most frequently used environmental isotopes for hydrological investigations of the soil system include isotopes of elements of the water molecule (1H (protium), 2H (deuterium), 16O and 18O) and that of element carbon (12C,and 13C) occurring in water as constituents of dissolved inorganic and organic compounds. Application of stable isotope ratios of hydrogen and oxygen in ground water is based primarily upon isotopic variations in precipitation.
The application of stable isotopes to hydrological cycling is based on the spatial and temporal variation of the isotope of elements of the water molecules. As water evaporates from the ocean surface, the lighter isotopes (1H and 16O) preferentially move to vapour phase because of the difference in vapour pressures and diffusion velocities and the resulting mass is depleted in lighter isotopes (2H and 18O). When water vapour condenses, these lighter isotopes get enriched in rain compared to the remaining vapour. The isotopic composition of precipitation is affected by season, latitude, altitude, amount and distance from the coast. The seasonal variation in temperature at a particular location generates strong seasonal variation in isotope composition of precipitation, with more depleted values occurring in the colder months than in summer. This variation acts as a tool to determine the time during the year when most recharge occurs in aquifers.
Isotopes can be used to identify water losses through evaporation from soil surface because the light isotopes (1H and 16O) evaporate more readily than the heavy isotopes (2H and 18O). The natural isotopic ratios of hydrogen (2H/1H) and oxygen (18O/16O), which are often expressed as delta units (δ2H and δ18O) in soil water, water vapour within a plant canopy and plant leaves can provide estimates of soil evaporation and plant transpiration. Such information will enable irrigation and land management practices to be developed to minimise soil evaporation and channel this water for crop production.
Soil organic matter
Natural abundance stable 13C isotope labelling is one of the few proven techniques available for the examination of soil C dynamics in naturally functioning ecosystems. Isotope ratio mass spectrometry (IRMS) is widely used to determine the difference in natural abundance of 13C between C3 and C4 vegetation which provides the basis for estimating the contribution of 13Cenriched C4 sources to soil organic matter (SOM) in ecosystems otherwise dominated by C3 vegetation. Following changes in land use from a C3 to C4 vegetation, bulk SOM δ13C values have been used to investigate organic matter(OM) turnover in soils, particle size fractions and soil aggregates. By using bulk and compound-specific IRMS, the spatiotemporal dynamics of whole C cycling, and that of specific biochemical components in the soil, can be determined.
To study the cycling of elements in the soil ecosystem, most studies rely on labelling experiments. The methodologies involving isotopes (among them 13C, 15N), are powerful research tools to assess the transfer and turnover of elements in the plant, the soil and the microbial compartments. Tracer techniques are also widely used to study i) the metabolic origin of an element within a compartment ii) the transformations and residence time of elements in a compartment, iii) the transfer of an element between compartments.
The labelling of plant shoots consists of offering 13CO2 (or 15NH4+ for studies on N rhizodeposition) to shoots for a certain period during plant growth. A combination of different tracer techniques (13C/15N) is an interesting field to make progress on understanding the plant-soil-microorganisms interactions and the fate of organic matter. Tracer techniques are used to perform foodweb analysis, especially to determine the fate of soil organic matter and the influence of different microbial communities.
Greenhouse gas production
Soil respiration is the largest component of ecosystem respiration and therefore, a key element in the carbon source/sink role especially for forest ecosystems. The ongoing increase in both atmospheric temperature and CO2 concentrations enhances the flux of CO2 from soil respiration. Promising new insights have been obtained from the use of stable carbon isotopes, where either: a) natural variability in the abundance of carbon isotopes in different compartments is examined; or b) a labelled 13CO2 signal is applied to compartments of the soil carbon cycle.
The natural abundance carbon isotope method has the potential of separating forest soil respiration components, although its applicability is limited to situations where overplanting from C4 to C3 plants occurred. On forest sites with an exclusive C3 vegetation history, the individual soil components must be physically isolated (disturbing the carbon cycle). In addition, natural δ13C signatures show high spatial variation allowing only for a rough partitioning of soil respiration.
The difficulty of relying on the existing C4 SOM can be overcome by the tank fumigation with 13C-depleted or 13C-enriched CO2. The newly formed organic material with depleted or enriched 13C signatures enables one to distinguish the root autotrophic respiration from heterotrophic respiration of litter, roots and SOM formed before labelling with natural abundance 13CO2.
A further important product of soil microbial processes is nitrous oxide (N2O), with its present concentration in the atmosphere of 350 ppbv, it is one of the important greenhouse gases accounting for approximately 5% of the total greenhouse effect. Atmospheric N2O along with carbon dioxide (CO2), methane (CH4), water vapour, etc absorbs and reflects back some of the thermal radiation emitted from the earth and increases its temperature.
Nitrous oxide also plays an important role in the destruction of the stratospheric ozone, which protects the earth from ultraviolet radiation from the sun.
The atmospheric concentration of N2O increased from 280-290 ppbv before the industrial revolution to 350 ppbv at present. Soil is considered to be one of the major contributors with 65% of the total global emission. Biological processes (denitrification, nitrification, dissimilatory nitrate reduction and assimilatory nitrate reduction) as well as abiological reactions (chemodenitrification) are possible mechanisms of N2O emission from the soil.
Denitrification occurs when nitrate is present in anaerobic microsites developed wherever microbial demand for O2 exceeds diffusion-mediated supply. This may well occur where O2 diffusion is impeded by water, either at the centres of soil aggregates or in water-saturated regions. Denitrification in soils also consumes N2O through the reduction of N2O to N2. Hence, this bacterial process may serve either as a source or as a sink of N2O. Nitrification also contributes to N2O emission following ammonium fertiliser or ammonia forming fertiliser addition to soils during the oxidation of NH4+ or NH2OH to NO3. The study of these processes is aided through the possibility of measuring the oxygen and nitrogen isotopes variations in N2O, NO3- or NH4+.
Conclusion
Although only a brief overview of the possibilities of stable isotope techniques has been presented here, the recent methodological and technical advances have greatly extended the possibilities for the application of stable isotopes to terrestrial ecology. A better understanding of soil processes is invaluable in predicting the future impacts of global environmental change on terrestrial ecosystems. New developments in instrumentation and methodology will assist in the further discovery of this important part of our ecosystem. n
Author
Dr Filip Volders
Is a graduate in Analytical Chemistry, University of Gent, and holds a PhD in Chemistry, University of Bristol. Post doctoral research in Italy, Germany (Max-Planck-Institute Biochemistry) as well as France completed his academic career. He has more than 15 years of experience in stable isotope analysis and works at present as Manager Productline IRMS for
Elementar Analysensysteme GmbH.
www.elementar.de
www.osedirectory.com/environmental.php
Published: 10th Mar 2011 in AWE International
