15N自然丰度法在陆地生态系统氮循环研究中的应用

随着氮沉降的不断增加以及人们对全球变化问题的日益关注, 稳定同位素技术在全球变化研究中得到广泛的应用。因为植物和土壤的氮同位素组成记录了氮循环影响因子的综合作用, 并且具有测量简单以及不受取样时间和空间限制的优点, 所以氮同位素自然丰度法被用于氮循环的研究中。该文从氮循环过程中植物和土壤的氮分馏入手, 总结国内外相关文献, 阐述了植物和土壤氮自然丰度在预测生态系统氮饱和和氮循环长期变化趋势中的应用; 总结了利用树轮δ ¹⁵N法研究氮循环过程中应该注意的事项以及目前尚未解决的问题。     

15N自然丰度法在生态系统氮素循环研究中的应用

稳定性同位素技术是现代生态学研究中的一门新兴技术,在生态学研究的诸多领域都展示了广阔的应用前景,其中,稳定性同位素＾１５Ｎ自然丰度法近年来在生态系统氮素循环研究中发挥了正在发挥着极为重要的作用,首先简述了自然生态系统氮素循环诸过程中的＾１５Ｎ同位素分馏机制,然后,在此基础上,综述了＾１５Ｎ自然丰度法的基本原理与方法,列举了近年 来此法在生物固氮及氮素转化过程研究中的一些应用实例,并预测了该方法在国     

15N自然丰度法在生态系统氮素循环研究中的应用

稳定性同位素技术是现代生态学研究中的一门新兴技术,在生态学研究的诸多领域都展示了广阔的应用前景。其中,稳定性同位素     

Gross mineralization and plant N uptake from animal manures under non-N limiting conditions, measured using 15N isotope dilution techniques

To utilise wisely the manure resource, a better understanding of the processes that control the breakdown of organic N to inorganic N (mineralization) is required. ¹⁵N isotope dilution techniques should allow estimates of plant N uptake and gross mineralization from organic manures under non-N limiting conditions to be made. In natural systems the study of organic nitrogen breakdown to inorganic nitrogen, mineralization, is confounded by the processes of nitrification, nitrate leaching, gaseous N losses and plant N uptake. The ¹⁵N isotope dilution approach allows measurement of gross mineralization independently of these processes. Greenhouse experiments were conducted to determine plant N uptake from organic manures under non-N limiting conditions using the soil pre-labelling isotope dilution approach. The soil was pre-labelled with ¹⁵N and maize plants were then grown on the control treatments (no organic amendment) or on the manure treatments. The principle is thus that the control crop has a ¹⁵N abundance which reflects the ¹⁵N status of the soil and the treatment crop has a ¹⁵N enrichment diluted by the contribution of mineralized unlabelled manure N. Using this technique, it was estimated that maize plants derived 17 and 34% of their N from sewage sludge and turkey manure, respectively. The soil pre-labelling isotope dilution approach allowed yield-independent estimation of nitrogen derived from manures under non-N limiting conditions. Estimates of gross N mineralization were made to determine the breakdown of manure under field conditions. Results suggested that there was a rapid mineralization of turkey manure N in the initial weeks after application, in the order of 50 kg N ha^?1, which tailed off in the following weeks. The technique suggested that the soil used in the study had an extremely low basal mineralization rate, and a high nitrification rate.     

基于15N示踪技术的植物-土壤系统氮循环研究进展

同位素示踪技术是指外源添加与生物体内的元素或物质完全共同运行的示踪物,用来指示生物体内某元素或物质变化过程的一种方法。利用氮稳定同位素示踪技术,能从本质上揭示生态学过程发生的机理,从而成为生态学科研工作十分重要的工具之一。对近年来15N示踪技术应用于土壤氮素固定、植物氮素营养和植物-土壤系统氮迁移的研究进展进行了综述,并对稳定氮同位素技术在解决相关生态学难题可能的前景和不足方面进行了展望。     

Exploration of inorganic C and N assimilation by soil microbes with time-of-flight secondary ion mass spectrometry

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect 13C and 15N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (12C14N-) and mass 27 (13C14N- and 12C15N-) ions sputtered with a Ga+ probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative 15N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with 15NO3-. We observed that the 15N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the 15N content of cells that have assimilated 15N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.     

What Have Stable Isotope Studies Revealed About the Nature and Mechanisms of N Saturation and Nitrate Leaching from Semi-Natural Catchments?

Various studies over the last 15 years have attempted to describe the processes of N retention, saturation and NO₃ ⁻ leaching in semi-natural ecosystems based on stable isotope studies. Forest ecologists and terrestrial biogeochemists have used ¹⁵N labelled NO₃ ⁻ and NH₄ ⁺ tracers to determine the fate of atmospheric deposition inputs of N to terrestrial ecosystems, with NO₃ ⁻ leaching to surface waters being a key output flux. Separate studies by aquatic ecologists have used similar isotope tracer methods to determine the fate and impacts of inorganic N species, leached from terrestrial ecosystems, on aquatic ecosystems, usually without reference to comparable terrestrial studies. A third group of isotopic studies has employed natural abundances of ¹⁵N and ¹⁸O in precipitation and surface water NO₃ ⁻ to determine the relative contributions of atmospheric and microbial sources. These three sets of results often appear to conflict with one another. Here we attempt to synthesize and reconcile the results of these differing approaches to identifying both the source and the fate of inorganic N in natural or semi-natural ecosystems, and identify future research priorities. We conclude that the results of different studies conform to a consistent conceptual model comprising: (1) rapid microbial turnover of atmospherically deposited NO₃ ⁻ at multiple biologically active locations within both terrestrial and aquatic ecosystems; (2) maximum retention and accumulation of N in carbon-rich ecosystems and (3) maximum leaching of NO₃ ⁻, most of which has been microbially cycled, from carbon-poor ecosystems exposed to elevated atmospheric N inputs.     

Water isotope analysis for tracing ecosystem processes: measurement techniques,ecological applications,and future challenges

水分是生态系统的重要因子, 水同位素自然示踪和人工标记是研究生态系统水循环过程的重要方法, 利用水同位素所具有的示踪、整合和指示等功能特征, 通过测量和分析生态系统中不同组分所含水分的氢氧同位素比值的变化情况, 可实现生态系统蒸散发的拆分、植物水分来源判定和叶片水同位素富集机理研究, 是研究生态系统水循环过程机理和生态学效应不可或缺的技术手段。该文首先简要回顾了生态系统水同位素发展和应用的历史, 在此基础上阐述了水同位素技术和方法在生态学研究热点领域应用的基本原理, 概述了水同位素在植物水分来源判定、蒸散发拆分、露水来源拆分、降水的水汽来源拆分以及 ¹⁷O-excess的研究进展, 并介绍了植物叶片水富集机理及基于稳定同位素的碳水耦合研究。最后, 指出了水同位素研究亟待解决的问题, 展望了水同位素应用的前沿方向, 旨在利用水同位素分析加深对生态系统的水分动态、植被格局和生理过程的理解。     

The use of natural abundance of nitrogen isotopes in plant physiology and ecology

Nitrogen stable isotope (¹⁵N, ¹⁴N) natural abundance has been much less used than carbon isotopes (¹³C, ¹²C) in plant physiology and ecology. Analytical problems, the lower fractional abundance of ¹⁵N than of ¹³C in the biosphere, the greater complexity of the N cycle relative to the C cycle, and smaller expressed discriminations in nature, are contributing factors. The major N pools, globally, have different isotope signatures: atmospheric N₂ is ¹⁵N-depleted relative to organic N (including sedimentary N), a situation resulting from a greater expressed discrimination in the organic N to N₂ (via denitrification) reaction than of diazotrophy during accumulation of the reduced N. Essentially all of the enzymes except nitrogenase which transform N compounds show discrimination against ¹⁵N, although for glutamine synthetase, and the amination of 2-oxoglutarate and pyruvate, this is only seen in terms of NH₄⁺ rather than the true substrate, NH₃. Discrimination is expressed in various N interconversions within plants, leading to substantial differences in δ¹⁵N (up to 12‰) among N compounds and macroscopic plant parts. N isotope fractionation during assimilation of exogenous combined N is often much lower than that expected from studies of isolated enzymes due to processes which show very little discrimination, such as limitation by transport through aqueous solution and membranes. Application of ¹⁵N/¹⁴N discrimination studies to plant ecology have concentrated largely on distinguishing diazotrophy from N supplied from combined N, based on the lower ¹⁵N/¹⁴N in diazotrophs due to the higher ¹⁵N/¹⁴N of combined N sources not being offset by fractionation during uptake. While potentially very useful, a number of pitfalls are discussed in its ecological use in both terrestrial and aquatic systems. N isotope discrimination is also useful in tracking N through food webs, and hence, back to combined N sources for plants.     

Stable isotopes as one of nature's ecological recorders

Analyses of the natural variation in stable isotopes of components of ecological systems have provided new insights into how these systems function across paleoecological to modern timescales and across a wide range of spatial scales. Isotope abundances of the molecules in biological materials and geochemical profiles are viewed as recorders that can be used to reconstruct ecological processes or to trace ecological activities. Here, we review key short-, medium- and long-term recording capacities of stable isotopes that are currently being applied to ecological questions. The melding of advances in genetics, biochemical profiling and spatial analysis with those in isotope analyses and modeling sophistication opens the door to an exciting future in ecological research.     

Nitrogen fixation,transfer and turnover in upland and lowland grass-clover swards,using15N isotope dilution

The stable isotope¹⁵N is particularly valuable in the field for measuring N fixation by isotope dilution. At the same time other soil-plant processes can be studied, including¹⁵N recovery, and nitrogen transfer between clover and grass. Three contrasting sites and soils were used in the present work: a lowland soil, an upland soil, and an upland peat. Nitrogen fixation varied from 12 gm^–2 on lowland soil to 2.7 gm^–2 on upland peat. Most N transfer occurred on upland soil (4.2 gm^–2) which, added to nitrogen fixed, made a total of 8.7 gm² input during summer 1985.¹⁵N recovery for the whole experiment was small, around 25%.Measurement of dead and dying leaves, stubble and roots, suggests that plant organ death is the first stage in N transfer from white clover to ryegrass, through the decomposer cycle. Decomposition was fastest on lowland soils, slowest on peat. On lowland soil this decomposer nitrogen is apparently subverted before transfer, probably by soil microbes.Variations in natural abundance of¹⁵N in plants were found in the two species on the different soils. These might be used to measure nitrogen fixation without adding isotope, but the need for many replicates and repeat samples would limit throughput.     

15N natural abundance of foliage and soil across boreal forests of Finland

This study presents the latitudinal variation (from 60° 30′ N to 68° 2′ N latitude) of natural abundances of ¹⁵N in the foliage, humus and soils of boreal forests of Finland. Our results clearly showed that N concentration of the foliage did not change significantly with latitudes but their ¹⁵N values were significantly higher in higher latitude sites relative to that of the mid and lower latitude sites, indicating the different forms of N uptake at the higher latitudes compared to the lower latitudes. We assume that the higher foliage δ¹⁵N values of the higher latitudes trees might be due to either more openness of N cycle (greater proportional N losses) in these latitudes compared to the sites of southern latitudes (lower N losses) or the differences in their mycorrhizal associations. Regression analysis showed that the temperature was the main factor influencing the ¹⁵N natural abundance of humus and soil of all forest ecosystems, both before and after clear-cut, whereas rainfall was the main controlling factor to the foliage ¹⁵N. Possible reasons behind the increasing δ¹⁵N natural abundances of plants and soils with increasing latitudes are discussed in this paper. The clear-cut did not show any specific trend on the ¹⁵N fractionation in humus and soil, i.e. both ¹⁵N-enrichment and -depletion occurred after clear-cut.     

Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel 15N2-DNA stable isotope probing (5N2-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from 15N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from 15N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that 15N2-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Organic vs. Conventional Grassland Management: Do 15N and 13C Isotopic Signatures of Hay and Soil Samples Differ?

Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the δ¹⁵N and δ¹³C isotopic composition of soil and hay samples of 21 organic and 34 conventional grasslands in two German regions. We also used Δδ¹⁵N (δ¹⁵N plant - δ¹⁵N soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances.Isotopic analyses revealed no significant differences in δ¹³C in hay and δ¹⁵N in both soil and hay between management types, but showed that δ¹³C abundances were significantly lower in soil of organic compared to conventional grasslands. Δδ¹⁵N values implied that management types did not substantially differ in nitrogen cycling. Only δ¹³C in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently used in practice.     

稳定性同位素技术与Keeling曲线法在陆地生态系统碳/水交换研究中的应用

稳定性同位素技术和Keeling曲线法是现代生态学研究的重要手段和方法之一。稳定性同位素能够整合生态系统复杂的生物学、生态学和生物地球化学过程在时间和空间尺度上对环境变化的响应。Keeling曲线法是以生物过程前后物质平衡理论为基础,将CO₂或H₂O的同位素组成(δD、δ¹³C或δ¹⁸O)与其对应浓度测量结合起来,将生态系统净碳通量区分为光合固定和呼吸释放通量,或将整个生态系统水分蒸散区分为植物蒸腾和土壤蒸发。在全球尺度上,稳定性同位素技术、Keeling曲线法与全球尺度陆地生态系统模型相结合,还可区分陆地生态系统和海洋生态系统对全球碳通量的贡献以及不同植被类型(C₃或C₄)在全球CO₂同化量中所占的比例。然而,生态系统的异质性使得稳定性同位素技术和Keeling曲线法从冠层尺度外推到生态系统、区域或全球尺度时存在有一定程度的不确定性。此外,取样时间、地点的选取也会影响最终的研究结果。尽管如此,随着分析手段的不断精确和研究方法的日趋完善,稳定性同位素技术和Keeling曲线法与其它测量方法(如微气象法)的有机结合将成为未来陆地生态系统碳/水交换研究的重要手段和方法之一。     

Advances in 15N-tracing experiments: new labelling and data analysis approaches

To obtain an in-depth understanding of soil nitrogen dynamics, it is necessary to quantify a variety of simultaneously occurring gross nitrogen transformation processes. In order to do so, most studies apply 15N in a disturbed soil-microbial-root system and quantify gross rates based on the principles of 15N isotope dilution. However, this approach has several shortcomings. First, studying disturbed soil provides only limited information on in situ soil nitrogen dynamics. Secondly, the analytical data analysis allows the quantification of total production and consumption rates of the labelled pool, but does not provide information on process-specific transformation rates. Combining in situ 15N isotope labelling over 1-2?weeks with numerical data analysis allows determining process-specific gross nitrogen transformations in undisturbed soils under field conditions in the presence of live roots and their associated microbial communities. This has the potential to increase our understanding of nitrogen dynamics in the soil environment.     

Ectomycorrhizal impacts on plant nitrogen nutrition: emerging isotopic patterns,latitudinal variation and hidden mechanisms

Ectomycorrhizal (EcM)‐mediated nitrogen (N) acquisition is one main strategy used by terrestrial plants to facilitate growth. Measurements of natural abundance nitrogen isotope ratios (denoted as δ¹⁵N relative to a standard) increasingly serve as integrative proxies for mycorrhiza‐mediated N acquisition due to biological fractionation processes that alter ¹⁵N:¹⁴N ratios. Current understanding of these processes is based on studies from high‐latitude ecosystems where plant productivity is largely limited by N availability. Much less is known about the cause and utility of ecosystem δ¹⁵N patterns in the tropics. Using structural equation models, model selection and isotope mass balance we assessed relationships among co‐occurring soil, mycorrhizal plants and fungal N pools measured from 40 high‐ and 9 low‐latitude ecosystems. At low latitudes ¹⁵N‐enrichment caused ecosystem components to significantly deviate from those in higher latitudes. Collectively, δ¹⁵N patterns suggested reduced N‐dependency and unique sources of EcM ¹⁵N‐enrichment under conditions of high N availability typical of the tropics. Understanding the role of mycorrhizae in global N cycles will require reevaluation of high‐latitude perspectives on fractionation sources that structure ecosystem δ¹⁵N patterns, as well as better integration of EcM function with biogeochemical theories pertaining to climate‐nutrient cycling relationships.     

Towards an understanding of the dynamics of compost N in the soil-plant-atmosphere system using 15N tracer

Aims

The principal aim of the present review is to synthesize and evaluate published information on the N fertilizer value of composts, and their effect on the utilization of conventional N fertilizers by crops.

Methods

We have examined the literature where the dynamics of N in the soil-plant-atmosphere continuum are traced using composts that were either artificially enriched in the ¹⁵N stable isotope (in units of atom % ¹⁵N excess) or had a natural ¹⁵N abundance (δ¹⁵N in units of ‰ or per mil) due to isotope discrimination processes that occur during composting. The methods used to produce artificially-enriched composts and to test uniformity of labelling are reviewed.

Results

Limited data show that composts are generally inferior sources of N for crops compared with their raw materials due to a lower N mineralization capacity. Immobilization of fertilizer N increases in compost-amended soils and may reduce recovery by a crop, but fertilizer N losses are reduced overall. However, co-application of compost and urea should be avoided due to the risk of increased NH₃ volatilization due to the action of compost-derived urease. High annual rates of compost application can exacerbate environmental problems including nitrate contamination of groundwater.

Conclusions

Efforts are required to improve the N fertilizer value of composts by minimizing NH₃ volatilization losses during composting. More attention should also be given to the use of the natural ¹⁵N abundance of compost as a tracer.     

Effect of nutritional condition on variation in δ13C and δ15N stable isotope values in Pumpkinseed sunfish (Lepomis gibbosus) fed different diets

Stable isotope analysis is frequently used to infer resource use in natural populations of fishes. Studies have examined factors, other than diet, that influence δ¹⁵N and δ¹³C including tissue-specific rates of equilibration and starvation. Most such studies completed under laboratory conditions tightly control food quantity and its isotopic composition, but it is also necessary to evaluate the influence of these factors under more natural conditions. Using pumpkinseed sunfish (Lepomis gibbosus) we evaluated whether restricted rations below minimum daily requirements affects tissue equilibration to a change in diet by holding fish on two treatments that often reflect divergent resource use in natural populations (pelagic zooplankton or littoral macroinvertebrates). Over 42 days, δ¹⁵N values increased while δ¹³C values did not change, additionally neither were related to diet treatment. Increased δ¹⁵N values were negatively related to body condition while δ¹³C values were not, indicating that stable isotope values were more affected by decreasing body condition than by diet. Additionally, δ¹⁵N values changed more in the blood and liver tissues than in white muscle tissue, indicating that restricting food availability had greater effects on tissues with greater metabolic activity. We hypothesize that stable isotope values of consumers are subject to a tissue-specific trade-off between sensitivity to changes in resource use and resistance to the effects of low resource availability. This trade-off may require consideration in stable isotope studies of wild populations facing periodic limitations of food availability.