Isotope applications to soil science at the University of Alberta — an historical perspective1

Abstract: For the past 70 yr, researchers in the Soil Science/Renewable Resources Department at the University of Alberta have used isotopes to study topics of ecological importance. This review highlights the soil isotope research conducted within our department over this time, including an historical overview of studies of interest. Analytical techniques and advances in instrumentation are discussed, focusing on the measurement of light stable isotope ratios (i.e., for C, H, N, S, and O) using isotope ratio mass spectrometry (IRMS). Early soil isotope work (1950–2000s) focused on agricultural soils and soil fertility issues. These studies included the use of radioactive isotopes such as 14C and 35S, and (or) artificially enriched stable isotopes including 15N-labelled fertilizers. More recently (2000–present), the scope of research widened to include natural-abundance stable isotope ratio studies as higher-sensitivity IRMS systems became more prevalent. Current isotope research topics include N biogeochemistry in natural and managed ecosystems, land management effects on greenhouse gas emissions, carbon cycling in northern landscapes, paleo-reconstruction in peatlands, carbon sequestration in boreal forests, and biodegradation of petroleum hydrocarbons. Further technological progress also enabled new techniques such as compound-specific IRMS analysis, including δ13C and δ2H measurements of soil n-alkanes and phospholipid fatty acids. In conclusion, current IRMS instrumentation presents unparalleled opportunities for multidisciplinary research to track carbon, plant nutrients, and pollutants as they move through soils.


Introduction
The Renewable Resources Department (formerly the Soil Science Department) at the University of Alberta just celebrated its 100th anniversary in 2019. For nearly 70 of the past 100 yr, soil scientists in that department have been using isotopes, the study of which is incredibly useful in ecological research. For those unfamiliar with isotopes, simply put, the atoms of most individual elements can exist in more than one form, whereas still exhibiting the same (or nearly the same) chemical properties. These different types of atoms of the same element are known as isotopes, and they differ from one another only by the number of neutrons in their nuclei. As a result, these isotopes have different masses (Michener and Lajtha 2007). For example, the three most common isotopes of carbon are carbon-12 ( 12 C), carbon-13 ( 13 C), and carbon-14 ( 14 C). All atoms of carbon contain six protons and six electrons, but the isotopes 12 C, 13 C, and 14 C contain 6, 7, and 8 neutrons each, with atomic mass numbers of 12, 13, and 14, respectively. Approximately 98.9% of the carbon found in nature is 12 C, 1.1% is 13 C, whereas a trace amount is 14 C. Both 12 C and 13 C are stable, which is to say they do not change over time, whereas 14 C is unstable, with a half-life of 5700 yr. It emits β − radiation, gradually decaying to 14 N (Garnier-Laplace and Roussel-Debet 2001).
Many ecological studies utilize the stable isotopes of light elements, in particular C, H, N, S, and O. Not only are these elements most frequently involved in biological processes, but the relative percent mass difference for light isotopes with a difference of only one or two atomic mass numbers is greater than for heavier elements (Michener and Lajtha 2007). For example, 13 C is 8.3% heavier than 12 C. This greater relative mass difference results in greater differences in isotope fractionation (accumulation or depletion of one particular isotope over another) due to naturally occurring mechanisms, thus making them easier to measure than heavy isotopes.
Although the approximate proportions of each naturally occurring isotope in the environment are known, the precise proportions of each isotope in a sample can vary for numerous reasons (Michener and Lajtha 2007). These can include natural enrichment/depletion, an increase/decrease in the relative concentration of one isotope (such as 13 C, 2 H, 15 N, 34 S, or 18 O) over other isotopes of the same element. Natural enrichment or depletion can occur through biochemical reactions; specifically, in biological systems, the product of any enzymatic reaction (e.g., photosynthesis, nitrification, and denitrification) is more depleted than the corresponding substrate, unless the entire substrate pool gets consumed (Michener and Lajtha 2007). In addition, artificial enrichment/depletion, such as the application of 15 N-labelled fertilizer allows tracking of the label as it travels through the soil-plant system. The relative amounts of isotopes in samples are typically measured by isotope ratio mass spectrometry (IRMS) after converting the target elements in the sample to simple gases such as CO 2 for C, H 2 for H, N 2 for N, SO 2 or SF 6 for S, and CO for O (Michener and Lajtha 2007). Results are generally reported as observed fractions of the rare isotope to the common isotope. The isotope ratio is expressed in delta notation, δ, in units of permil, ‰, relative to the isotope ratio of an internationally accepted standard. The light stable isotope ratios frequently measured by ecological researchers are therefore δ 13 C/ 12 C, δ 2 H/ 1 H, δ 15 N/ 14 N, δ 34 S/ 32 S, and δ 18 O/ 16 O.
The isotopic ratios of a sample can be used to determine where certain elements in the sample originated from. IRMS has become an important tool in environmental research, with numerous applications (Chabbi and Rumpel 2012). Researchers can use δ 13 C isotope ratio data to trace carbon fluxes in the terrestrial carbon cycle (Brüeggemann et al. 2011). In the above-mentioned case of labelled 15 N fertilizer application, IRMS can be used to trace where that fertilizer travels, how much is taken up by the target crop, and how much is lost to other parts of the environment (Malhi and Nyborg 1983). More recent developments in soil isotope techniques include the use of compound-specific IRMS analyses to track δ 13 C ratios in soil microbial biomarkers, i.e., phospholipid fatty acids (PLFAs) (Glaser 2005;Watzinger 2015). The possibilities for isotope ratio research in soil science are endless, and numerous examples of IRMS applications in soil science will be discussed in this review, including the use of natural abundance and enrichment techniques.
This review focuses on isotope work conducted within the Soil Science Department from the 1950s to 2019 at the University of Alberta, and within the Renewable Resources Department following the merger of the Soil Science and Forestry Departments in 1994. Figure 1 shows the number of articles published using soil isotopes by members of our department over this time span. The list of references used to prepare this figure was compiled from a combination of search results for soil isotope papers from the University of Alberta using the Web of Science and Scopus databases (up until 15 July 2019), along with internal departmental records. Although every effort has been made to include mention of all researchers within the department who have conducted studies using soil isotopes over the last 70 yr, it is possible that some contributors may have been inadvertently left out of this discussion. Additionally, to limit the breadth of this review to a manageable scope, this article focuses on members of the University of Alberta's Soil Science/Renewable Resources Department. However, other researchers at this university have and continue to conduct environmental research using stable isotopes, including our colleagues in the Department of Chemistry, the Department of Earth and Atmospheric Sciences, and the Department of Agricultural, Food, and Nutritional Sciences.
Although this review focuses on work at the University of Alberta, several other universities in Canada also have long histories of soil and other environmental stable isotope research. The Ján Veizer Stable Isotope Laboratory (formerly the G.G. Hatch Lab) at the University of Ottawa has been at the forefront of light stable isotope research and education for many years, as has the University of Saskatchewan. The Universities of Calgary, Lethbridge, British Columbia, and Waterloo are all home to researchers involved in soil stable isotopes, and this is by no means a comprehensive list.

Radioactive isotope work
Although the main focus of this article is on light stable isotope research in soil science at the University of Alberta, such a discussion would not be complete without mentioning the numerous soil studies conducted using radioactive isotopes. Below is a brief overview of such research from members of the Soil Science/Renewable Resources Departments.

S
Isotopes have been used in soil science research at University of Alberta since the 1950s. The earliest such studies were conducted by C.F. Bentley and D.B. Scott, along with Bentley's graduate students D.V. Cormack and D.J. Hoff (Cormack et al. 1951;Bentley et al. 1955). These researchers used radioactive 35 S-labelled fertilizer to trace uptake in legume and barley crops, using a Geiger-Müller tube to count activity. The test crops were grown at the Breton Plots, a research facility established in 1929 that is still in use by the University of Alberta today. Further, similar research was later continued by Pawluk and Bentley (1964). Additionally, R.A. Drijber, a Ph.D. student supervised by W.B. McGill who graduated in 1993, used 35 S to study the use of sulfonolipids as biomarkers for Cytophaga and Flexibacter bacteria in soils McGill 1994a, 1994b).

C
Radioactive 14 C was used by several soil science researchers at the University of Alberta, the first of whom was W.B. McGill. McGill received his Ph.D. from the University of Saskatchewan in 1971, and along with J.A. Shields and E.A. Paul (McGill et al. 1975), used 14 C and 15 N to trace metabolic pathways in soil microbes. In these studies, 14 C radioactivity was measured, and 15 N content was determined using an Atlas Model GD 150 mass spectrometer. McGill later came to the University of Alberta and continued his research with soil isotopes, much of which will be discussed in the stable isotopes section of this article. However, his research still included radioactive carbon-14. For example, McGill's Ph.D. student C.M. Monreal used 14 C to study cystine cycling in soil (Monreal and McGill 1989a, 1989b, 1989c, 1989d.
N.G. Juma is another soil scientist who used 14 C in his research, along with his graduate students G.D. Dinwoodie and J.G. Xu. Dinwoodie and Juma used 14 C to study carbon dynamics in barley plots, using a Minaxi β Tri-Carb 4000 series scintillation counter to measure 14 C activity (Dinwoodie and Juma 1988). Later, Xu and Juma used 14 C to study kinetics in root systems (Xu and Juma 1993, 1995. We will see more of Juma's contributions to soil isotope research later in this paper.

P and 137 Cs
Although studies including soil phosphorous data were frequently published by University of Alberta researchers, a few using radioactive 32 P were also conducted. J.A. Robertson and his student P.K. Omanwar used 32 P to study soil exchange kinetics in the 1970s (Omanwar and Robertson 1971). Lastly, the work of M.J. Dudas and C.P. Maulé involving radioactive cesium is worth mentioning. These authors used 137 Cs, produced by nuclear testing in the 1950s and 1960s and deposited in the environment, to trace soil erosion (Maulé and Dudas 1989). These studies followed earlier work by E. de Jong et al. at the University of Saskatchewan (De Jong et al. 1982).

Stable isotope work
The earliest soil stable isotope research conducted by members of the University of Alberta Soil Science Department was published in 1968 by R.P. Wellman, F.D. Cooke, and H.R. Krouse (Wellman et al. 1968). In this study, the authors measured the fractionation of 15 N/ 14 N during the microbial reduction of nitrate/nitrite to N 2 .
Other early examples include the previously mentioned work of W.B. McGill, E.A. Paul,et al. using 15 N in conjunction with 14 C to study soil microbial pathways (McGill et al. 1975;McGill and Paul 1976). 15 N stable isotope analysis would become a major theme for several researchers at the University of Alberta. M. Nyborg, a professor in the Department of Soil Science for many years, is one such example, and his isotope-focused research examined soil fertility and fertilizer N and S uptake by plants. Together with S.S. Malhi, who received his Ph.D. in 1978 while studying under M. Nyborg, the two authors published several 15 N papers over the years. In one study, they used 15 N-labelled fertilizer, applied in the fall, to determine how much N was lost due to denitrification, immobilization, and leaching over the winter (Malhi and Nyborg 1983). They determined that fall application of fertilizer resulted in large overwinter losses, which were due to denitrification rather than leaching. Nyborg published further research on this topic with D.J. Heaney (Heaney and Nyborg 1988;Heaney et al. 1992).
In addition to application timing and fertilizer recovery, Malhi and Nyborg's 15 N fertilizer research included studies of fertilizer placement methods. They also used different types of fertilizer 15 N sources, such as nitrate and urea, and worked with several other students and researchers including E.D. Solberg, R.C Izaurralde, M. Zhang, and R. Pradhan (Malhi et al. 1989;Nyborg et al. 1990;Malhi and Nyborg 1991;Pradhan et al. 1998;Zhang et al. 2000). This research typically involved the application of isotopically enriched 15 N fertilizer to soil systems and subsequent analysis by IRMS. Much of these analyses were conducted using a Micromass 602C Mass Spectrometer, which was acquired by the Soil Science Department in 1975. This was later replaced by a Stable Isotope Ratio Analyzer (SIRA) 10 from VG Isogas in the UK, acquired in 1987, and often used coupled to an Automatic Nitrogen Analyzer 1500. N.G. Juma, another long-serving professor at the Soil Science Department at the University of Alberta, used 15 N in his soil research. He studied C and N transformation in the soil, developing models of nutrient cycling. Some of his earlier publications involving isotopes, conducted in collaboration with the University of Saskatchewan, include studies of soil N mineralization kinetics and N immobilization in the presence of nitrification inhibitors (Juma and Paul 1983;Juma et al. 1984). P.M. Rutherford, a Ph.D. student of N.G. Juma, used 15 Nenriched urea to compare plant-soil N dynamics in Black Chernozemic and Gray Luvisolic soils cropped to barley (Rutherford and Juma 1989). In subsequent work, Rutherford and Juma (1992) used 15 N (and 14 C) to show that protozoan grazing on soil bacteria induced more significant mineralization of bacterial N and C in coarse-textured soil than in medium-and fine-textured soils. Working with Juma, M.Sc. student K. Haugen-Kozyra studied N dynamics in barley-soil systems under conventional and zero tillage. More 15 N-enriched urea was converted into organic N under zero tillage than conventional tillage suggesting better conservation of N in the former system (Haugen-Kozyra et al. 1993).
Much of the stable isotope ratio research conducted in the 1970s and 1980s by members of the Soil Science Department was done using 15 N. However, G.A. Spiers' research in clay mineral weathering made use of 18 O. Spiers was supervised by M.J. Dudas and S. Pawluk for his M.Sc. (1982) and Ph.D. (1990), respectively. These researchers, in collaboration with K. Muehlenbachs from the Geology Department (later the Earth and Atmospheric Sciences Department), used the BrF 5 method to isolate and measure δ 18 O in clay separates isolated from soil samples (Spiers et al. 1985).
Some further discussion of W.B. McGill's research as a professor in the Soil Science/Renewable Resources Department is warranted. McGill's work can broadly be described in two categories; soil biogeochemistry, and the fate and transport of organic compounds in soil. In 1985, along with colleagues from Duke University and Oregon State University, he published a study using 15 N to trace alder-fixed N in conifers (Binkley et al. 1985). Notably, in this research, the authors measured naturalabundance 15 N in their samples. Up until this point, most isotope ratio studies at the University of Alberta used artificial 15 N enrichment to trace soil N. Another illustrative example of McGill's use of 15 N in soil research is the work conducted by his Ph.D. student D.C. Jans-Hammermeister studying soil-plant dynamics in barley crops by green manuring with field pea (Jans-Hammermeister et al. 1994).
The works of one additional soil researcher from the University of Alberta, D.J. Pluth, are worth mentioning. In collaboration with McGill, Pluth and his Ph.D. student, G.E. Nason, as well as R.T. Hardin from the Department of Animal Science, studied foliar N dynamics in Douglas-fir with 15 N isotopically enriched ammonium nitrate and urea (Nason et al. 1990). Also, Pluth and S.E. Macdonald, the current chair of the Renewable Resources Department, co-supervised Ph.D. student A.G. Mugasha, who studied at the University of Alberta while on leave from the Tanzania Forestry Research Institute. Mugasha and Pluth used 15 N-labelled urea fertilizer to study soil and tree uptake patterns in tamarack and black spruce stands Pluth 1994a, 1994b). Pluth also collaborated with researchers from the Swedish University of Agricultural Sciences and Forestry Canada to characterize 15 N fractionation in soils and Scots pine (Nômmik et al. 1994). Although the IRMS analysis for this publication was not conducted at the University of Alberta, it is another of the rare, early examples of natural-abundance 15 N isotope ratio studies.

Recent and current soil isotope research (early 2000s-present)
After a lull in publications related to soil isotope research in the late 1990s, new faculty hires, starting with S.X. Chang and S.A. Quideau in the early 2000s, generated a fresh wave of research at the University of Alberta, which resulted in a steady increase in publications since then (Fig. 1). In addition, isotope research evolved from its early focus on soil fertility to encompass broader environmental issues. Current research within the Department of Renewable Resources that utilize isotopic tools includes a wide range of subjects such as: N biogeochemistry in natural and managed ecosystems (S.X. Chang, M.D. MacKenzie, and S.A. Quideau); land management effects on greenhouse gas emissions (G. Hernandez-Ramirez); carbon cycling in northern landscapes (D. Olefeldt); paleo-reconstruction in peatlands (W. Shotyk); carbon sequestration in boreal forests (S.X. Chang and S.A. Quideau); and biodegradation of petroleum hydrocarbons (T. Siddique). In conjunction with a change in focus, research has shifted from using radioactive to stable isotopes, and recent projects have taken advantage of technological advances to measure compound-specific and position-specific isotopic values.

Nitrogen isotopes
Enriched 15 N work Some of our department's more current work has continued to take advantage of 15 N-enriched fertilizers to trace the source of N for plant uptake. For example, in the group led by S.X. Chang, 15 NH 4 NO 3 and NH 4 15 NO 3 were used to study the preferential uptake of NH 4 + vs. NO 3 − by trembling aspen and hybrid aspen seedlings (Choi et al. 2005a). The uptake and recovery of 15 N per tree was higher for the hybrid aspen than for the trembling aspen, and both species exhibited a higher recovery of 15 NH 4 + than 15 NO 3 − , which goes against the common assumption that deciduous tree species are better adapted to nitrate than ammonium nutrition. The 15 N pool dilution method has been another commonly used isotopic tool in S.X. Chang and S.A. Quideau's groups to quantify gross N transformation rates in a range of soil environments and land uses, including forestry and agriculture, as well as in landscapes undergoing reclamation or other land use changes (McMillan et al. 2007;Lang et al. 2010Lang et al. , 2019Cheng et al. 2012Cheng et al. , 2019. In this method, labelled inorganic ammonium and nitrate are applied to the soil to quantify gross ammonification and nitrification rates. In addition, 15 N-enriched litter obtained from plants grown with 15 N-labelled fertilizer can be used to quantify mineralization rates. C. Norris (Ph.D. with S.A. Quideau) used this approach to quantify N cycling in natural and reclaimed soils (Norris et al. 2018).

Natural 15 N abundance
In soils, natural variations in N stable isotopes result from diverging δ 15 N values of external N sources and internal ecosystem transformations. Many of the internal, microbial-driven N processes (e.g., nitrification, denitrification, and immobilization) result in measurable discrimination between substrate and product, where the product is more depleted in N than the substrate. This causes a progressive 15 N enrichment of the residual soil N pool, which together with plant δ 15 N values, can be used as an index of the ecosystem N status (Fang et al. 2011). M.D. MacKenzie, a soil scientist studying fertility and N cycling related to disturbance in forest ecosystems, used this natural variation in N stable isotopes to follow the ecological response to N deposition of reclaimed forest stands in the oil sands region of Alberta (Hemsley et al. 2019). The soil, foliar, and root pools of jack pine stands were more enriched in δ 15 N compared with the trembling aspen stands. Hence in this study, isotopic analysis provided one of the key indicators that pine was more sensitive to the additional N deposition and that these ecosystems may be approaching saturation. Similarly, many of the studies in S.X. Chang's group have taken advantage of δ 15 N measurements in foliage and woody tissues to evaluate the effects of land use on plant nutrition. Some of these studies have (i) quantified the effects of soil compaction and forest floor removal on N cycling in lodgepole pine and Douglas-fir stands (Choi et al. 2005b), and trembling aspen (Tan et al. 2006); (ii) explored the relationship between N deposition and tree ring chemistry of jack pine and trembling aspen stands (Jung et al. 2013); and (iii) studied the relationship between soil nitrification and foliar δ 15 N in trembling aspen and jack pine forests of different stand age (Hu et al. 2014).
Variations in soil N isotopes linked to changes in environmental conditions may be too small or short lived to be useful as diagnostic tools. In contrast, one systematic variation has been linked to trophic levels, with the lower positions being relatively less enriched than the higher trophic levels, and this consequently has been recognized as a powerful tool for reconstructing trophic relationships in soil food webs (Scheu and Falca 2000). Irma Diaz (Ph.D. in S.A. Quideau's group) used the δ 15 N values of soil mite bodies to position them along a trophic gradient in the forest floors of coniferous and deciduous stands of western Canadian boreal mixedwood forest. In addition to mature (control) stands, she also studied the potential effect of clear-cutting on these trophic guilds (Diaz-Aguilar and Quideau 2013). The difference of δ 15 N values in mites placed them in three distinct trophic guilds: detritivores, omnivores, and predators. Furthermore, the trophic position of these forest floor mites stayed constant regardless of their habitat.

Optical spectroscopic techniques
Globally, nitrous oxide represents more than half of the agriculture-derived greenhouse gas emissions. However, wide spatial and temporal variation of soil N 2 O fluxes brings high uncertainty to estimates of rates and sources. Two formation pathways for N 2 O have been identified, denitrification by anaerobic bacteria, or as a by-product of aerobic nitrification (Baggs 2008). The development of cavity ring-down spectroscopy, which allows measurements of site-specific isotopic composition as well as bulk δ 15 N in N 2 O, now allows separation of the two pathways. Namely, the difference in δ 15 N values between the central and terminal positions of N 2 O can be used to differentiate between the nitrification (mean value of 33‰) and denitrification (mean value of 0‰) pathway (Ibraim et al. 2019). This approach is currently in use by G. Hernandez-Ramirez to determine the effects of various organic amendments on nitrous oxide emissions from agricultural soils (unpublished results).

Carbon isotopes
Natural abundance 13 C Elucidating humification processes responsible for the transformation of plant residues into soil organic matter is a key to understanding global carbon biogeochemistry and the controls of carbon accumulation in soils. Peatlands and other organic soils are unique in that their organic matter is not stabilized by interactions with the soil mineral matrix as is the case with other soil types. Yet, peatland organic matter can be preserved for centuries and sometimes millennia, and as such, can serve as a valuable archive of past environmental conditions. W. Shotyk, a soil and water geochemist interested in the cycling of trace metals, has been using sphagnum moss and peat bogs to reconstruct their natural and anthropogenic sources to the atmosphere. Numerous experimental approaches and instrumental tools have been proposed and utilized to study peat formation processes. In regard to the specific use of light stable isotopes, researchers in W. Shotyk's group have investigated the value of δ 13 C and δ 15 N measurements as indices of peat humification extent (Zaccone et al. 2018).
One of the most common themes in the use of the natural abundance 13 C technique is to use δ 13 C data from plant samples to infer the effect of different factors (management or edaphic) on plant water use efficiency (see for example, the following work from S.X. Chang: Choi et al. 2005bChoi et al. , 2007Tan et al. 2006;Matsushima et al. 2014). Additionally, as soil organic matter becomes progressively enriched in 13 C with increasing humification, its isotopic composition can be a useful tool to explore decomposition processes and the extent of microbial processing (Quideau et al. 2003). This approach has been successfully applied to the study of organic matter processes in reclaimed (Turcotte et al. 2009), arboreal (Enloe et al. 2010), riparian (Card et al. 2010), and agricultural (Kipps 2015) soils, and harvesting effects in forest soils (Hannam et al. 2005;Sewell 2018).
Enriched 13 C Carbon dioxide ( 13 CO 2 ) pulse labelling of plants is a powerful method to trace the fate of carbon fixed by photosynthesis. This technique was used by C. Arevalo (Ph.D. with S.X. Chang), who measured 13 C incorporation in different plant tissues (leaves, stems, and roots) of two hybrid poplar clones, and studied how that incorporation was affected by plant age (Arevalo et al. 2010). In addition, C. Norris and S.A. Quideau combined 13 C-pulse labelling with characterization by solid-state 13 C nuclear magnetic resonance (NMR) spectroscopy to track the incorporation of the 13 C label into aspen leaf and root macromolecules (Norris et al. 2012). This work was conducted in conjunction with R.E. Wasylishen's solid-state NMR research group in the Department of Chemistry. Combining C and O isotope composition ( 13 C, 14 C, and 18 O) to molecular characterization by NMR allowed the quantitative comparison of cellulose preparation techniques (Gaudinski et al. 2005). Lastly, combining soil physical fractionation and isotopic and NMR analyses allowed Norris et al. (2011) to assess how soil carbon stabilization under pine vegetation varied along a climosequence from Saskatchewan to Manitoba. D. Olefeldt works on carbon cycling in boreal to arctic landscapes, with a focus on impacts of permafrost thaw, wildfire, droughts, and human activities on carbon storage. Northern permafrost peatlands store more carbon per unit area than any other biome on earth. Increased air temperature, and associated increased fire frequency, are both contributing to the rapid thawing of these permafrost soils, with the concomitant release of carbon to the atmosphere and possibly to aquatic systems. By combining measurements of 14 C of dissolved organic carbon and CO 2 released from northern permafrost peatlands to 13 C-CO 2 analyses, D. Olefeldt and his group were able to co-currently determine the age and sources of CO 2 to streams and to differentiate between CO 2 released from weathering vs. soil organic matter degradation (Burd et al. 2018;Estop-Aragonés et al. 2018). In addition, 13 C-CH 4 chamber measurements were used to assess whether methane was produced via acetoclastic or hydrogenotrophic pathways in wetland soils of different permafrost conditions and from thermokarst ponds in peatlands (Hutchins et al. 2020). Lastly, 13 Clabelled glucose was added to peat during anaerobic incubations to test hypotheses of primingagain to study whether peat following permafrost thaw is vulnerable to rapid decomposition (unpublished results).

Compound specific isotopic analysis (CSIA)
Stable isotope probing (SIP) of microbial PLFAs, lipids found in microbial membranes, is a method that tracks the incorporation of labelled substrates into distinct microbial groups found in the soil environment. The label can be added as 13 C-enriched CO 2 and followed from root exudates into rhizospheric communities (Béasse 2012). The label can also be added as a simple substrate (e.g., 13 C-enriched glucose) or as more complex 13 C-enriched plant materials. For instance, Norris et al. (2016) used double-labelled aspen litter ( 13 C and 15 N) to compare soil microbial activity and function in aspen and spruce stands, which are typical of the boreal mixedwood landscape of western Canada. Similarly, Lloret and Quideau (2015) incubated soils sampled under both aspen and spruce to study how microbial communities processed different 13 C-labelled substrates (glucose, leaves, and roots). Carbon assimilation by microbes was tracked by isotope probing of PLFAs, and measurement of evolved 13 C-CO 2 allowed quantification of the percent of CO 2 coming from each added substrate. Incubation of 13 C-labelled aspen litter in spruce soils mimics the effects of anticipated future vegetation shifts, and results suggested that shifting from spruce to aspen in the boreal forest may increase microbedriven carbon stabilization. More recently, Lejoly et al. (2020) used a similar approach to compare carbon processing in natural and reclaimed sandy soils.
More than half of the total carbon in forest soils is stored below a depth of 20 cm. Although forest floor carbon is known to turn over quite rapidly, the stability of carbon stored deeper in forest soil profiles still remains uncertain. Quideau et al. (2018) compared carbon stability in Luvisolic and Podzolic B horizons by measuring total soil respiration and explored the potential of measuring natural 13 C abundance of the respired CO 2 to differentiate between sources. The natural abundance 13 C composition of microbial PLFAs was observed to differ between rhizosphere and bulk soil; namely, rhizosphere PLFAs showed 13 C depletion compared with bulk forest floor, indicating that rhizosphere microbes were accessing more recently fixed carbon than in bulk soil (Thacker 2018). Lastly, a multi-isotope approach may be the best strategy to constrain soil processes and elemental fluxes. For instance, Paul and Quideau (2020) used 13 C and 2 H analyses of n-alkanes to contrast humification processes in reconstructed, peat-dominated soils, and natural, native forest soils.
T. Siddique, an environmental soil chemist in the Department of Renewable Resources, has been using DNA-SIP to identify the specific microorganisms involved in the biodegradation of petroleum hydrocarbons. In particular, labelled toluene was used as a model compound for BTEX in biodegradation study under methanogenic conditions (Zamir et al. 2012). Finally, the biodegradation of naphtanic acids, contaminants found in tailing ponds of Northern Alberta's oil sands, was followed using 13 C-labelled surrogates. More specifically, 13 C-PLFA analysis allowed the authors to differentiate microbial uptake of 13 C-enriched surrogates and 13 C-depleted biogenic methane (Ahad et al. 2018).

Early instrumentation
In 1975, the Soil Science Department at the University of Alberta obtained its first IRMS system, a Micromass 602 Mass Spectrometer. Figure 2a shows a photo of this instrument in operation by technician C. Nguyen in 1985. Early IRMS instruments generally operated as dual-inlet systems, alternating between the analysis of sample gas and reference gas. Such systems are still in use today, as the dual inlet approach has several advantages, including high precision (Michener and Lajtha 2007) and low consumption rates of reference gases. However, continuous-flow IRMS systems eventually grew in popularity. They permitted the analysis of sample gases like CO 2 and N 2 immediately after preparation, such as from the outlet of a combustion elemental analyzer (i.e., continuous flow elemental analyzerisotope ratio mass spectrometry, EA-IRMS). This allowed for the rapid, automated measurement of isotope ratios from weighed solid samples, including soils and plant material. The Micromass unit in the Soil Science Department was later replaced in 1987 by a VG Isogas SIRA 10 Continuous Flow IRMS, interfaced to a Carlo-Erba NA1500 Strumentazione combustion elemental analyzer. These instruments were used for δ 13 C and δ 15 N stable isotope ratio analysis.
As mentioned previously, early studies in soil isotope ratios were primarily conducted using samples isotopically enriched in 15 N and (or) 13 C. As instruments were designed and built with higher sensitivity and precision, such research began to include measurements on natural-abundance samples, with no isotopic enrichment. In 2003, IRMS capabilities within the Renewable Resources Department continued to expand with the acquisition of a ThermoFinnigan Delta+ Advantage IRMS purchased from Isomass Scientific. This unit was a workhorse in the Soil Science/Renewable Resources Department for many years. It was interfaced to a Costech 4010 Elemental Analyzer through a ConFlo III continuous-flow system for EA-IRMS. Like the VG Isogas unit, this was used for bulk δ 13 C and δ 15 N analysis of soils, plant material, and other ecological samples, but the higher sensitivity of the ThermoFinnigan instrument allowed for other applications as well. It was also interfaced to a gas chromatography-combustion (GC-C) unit, which, for the first time, enabled researchers at the University of Alberta to conduct compound-specific δ 13 C isotope ratio analysis of soil extracts. Though no longer used for EA-IRMS or GC-C-IRMS, the ThermoFinnigan unit is still used today, interfaced to a GasBench II system for δ 13 C analysis of CO 2 .

Current configuration
In 2016, the acquisition of a suite of new instruments and peripherals at the Renewable Resources Department established the Stable Isotope Facility for Ecosystem Research (SIFER). A photograph of this facility is shown in Fig. 2b. This laboratory is currently equipped with three isotope ratio mass spectrometers. The first is the ThermoFinnigan Delta+ Advantage IRMS from 2003 discussed earlier. In 2016, this IRMS was connected to a total organic carbon (TOC) interface from Isomass Scientific which, along with an OI 1030W TOC analyzer, permits the δ 13 C analysis of total or dissolved organic or inorganic carbon in water samples. The rest of the systems currently used in SIFER were all obtained in 2016. For bulk EA-IRMS analysis, we use a Thermo Scientific Delta V Advantage IRMS with ConFlo IV continuous-flow interface. A Thermo Scientific Flash 2000 HT Plus Elemental Analyzer Unit completes this system. When operated in combustion mode, this allows for bulk δ 13 C and δ 15 N analysis in solid samples, much like with the previous ThermoFinnigan and Costech systems, but with better sensitivity. However, when used in pyrolysis mode, bulk analysis of δ 2 H and δ 18 O in solid samples is also possible. A liquid autosampler and injection system permits δ 2 H and δ 18 O analysis of water samples as well.
The last IRMS system currently housed in SIFER is our highest sensitivity instrument, a Thermo Scientific Delta V Plus IRMS. This instrument is equipped with two different chromatography systems and is used for compound-specific isotope ratio studies. A Thermo Scientific Trace 1310 GC and GC Isolink II enable compound-specific analysis of GC samples for δ 13 C and δ 15 N (combustion mode), or δ 2 H (high-temperature conversion mode). This system has numerous potential applications, allowing isotope ratio analysis of not just bulk samples, but individual chemical species within each sample. So far, we in SIFER have analyzed δ 13 C in PLFA extracts from soil samples, and δ 2 H in n-alkanes in soil extracts. For liquid chromatography samples, a Thermo Scientific Ultimate 3000 LC system with LC Isolink permits compound-specific δ 13 C analysis in aqueous matrices. Finally, for greenhouse gas isotope ratio studies, SIFER is equipped with a Picarro G2201-i cavity ring down spectroscopy system. This instrument allows for the rapid analysis of δ 13 C in CO 2 and CH 4 in gas samples and is field portable.

IRMS technicians
Numerous technicians and support staff members in the Soil Science/Renewable Resources Department were responsible for the operation and maintenance of IRMS instrumentation at the University of Alberta, including C. Figueiredo, C. Nguyen, and J. Khatkar. A. Harms, currently the manager of the department's Natural Resources Analytical Laboratory, has been with the department since 1997 and continues to operate and maintain these systems today.

Challenges and Opportunities
Major advances in instrumentation, especially with regard to the continuous-flow isotope ratio mass spectrometers (CF-IRMS) interfaced to a range of peripherals have allowed high-precision measurements of increasingly small sample sizes (Crotty et al. 2013). Coupling of CF-IRMS to gas and liquid chromatographs in the mid-80s opened the door to the isotopic analysis of individual molecules (i.e., CSIA). Analyses of some metabolites that are well suited to gas chromatography, such as δ 13 C values of soil microbial phospholipids, are now mostly routine in isotope laboratories. Analytical precision while measuring natural isotopic abundance in individual PLFAs has improved to the point where it is possible to differentiate between carbon sources with little isotopic separation. Such was the case for PLFAs extracted from rhizospheric and bulk soil samples (Thacker 2018). Other emerging applications include δ 15 N analysis in individual amino acids (Ohkouchi et al. 2017) and combined analysis of multiple isotopes, such as δ 13 C and δ 2 H measurements in soil n-alkanes (Paul and Quideau 2020). CSIA is particularly well suited to soil metabolomics, and it is a powerful tool to trace natural metabolites and pollutants alike. Newer technology is now in place to follow molecules that are best analyzed with liquid chromatography, such as soil carbohydrates and amino acids. However, while this analytical approach possesses tremendous potential, applications to date have been limited, mostly due to the cost and the rarity of CSIA instrumentation dedicated to environmental research.
The development of non-mass spectrometry approaches (i.e., optical spectroscopic techniques) has literally brought isotopic analyses to the field. Compared with traditional IRMS analyses, spectroscopic instruments present some advantages, including lower cost, ease of use, and maybe more importantly portability. These infrared (IR) absorption laser spectrometers utilize either the more sensitive (but less robust) mid-IR technology or the most commonly used, cheaper near-IR technology. Combined with cavity ring down technology, near-IR absorption spectroscopy lasers are particularly well adapted to the measurements of greenhouse gases under various field conditions, including remote locations.
Technical advances in the area of isotope geochemistry have been nothing short of phenomenal since the creation of the first isotope mass spectrometers in the first half of the 20th century. The first published work on carbon stable isotopes, using what was considered at the time to be a "mass spectrometer of high sensitivity and high resolving power" quoted a precision of 0.5% for the 12 C/ 13 C ratio (Nier and Gulbransen 1939). By contrast, modern IRMS systems can easily reach δ 13 C/ 12 C precisions of ±0.2‰, and many systems routinely achieve precisions much better than that. Today, isotopes of light elements (C, H, N, O, and S) provide environmental science researchers the means to trace the origins and track the fates of individual metabolites, nutrients, and chemical pollutants as they move through soils, plants, food webs, soil and surface waters, and the atmosphere.