15N2 as a tracer of biological N2 fixation: A 75-year retrospective

doi:10.1016/j.soilbio.2016.12.010

Soil Biology and Biochemistry

Volume 106, March 2017, Pages 36-50

https://doi.org/10.1016/j.soilbio.2016.12.010 Get rights and content

Highlights

•
¹⁵N₂ is the only direct method for quantifying biological N₂ fixation (BNF).
•
The central role of ¹⁵N₂ in studies of BNF over the past 75 years is reviewed.
•
Indirect ¹⁵N methods for estimating BNF should be verified by the ¹⁵N₂ method.
•
Stable isotope probing with ¹⁵N₂ can identify uncultured diazotrophs.
•
The role of BNF as a source of reactive N and N₂O requires further study.

Abstract

¹⁵N₂ has played a crucial role in fundamental studies of biological N₂ fixation. However, due to operational constraints, it has more often served as a qualitative rather than a quantitative tracer of biologically-fixed N (BFN). Therefore indirect methods based either on ¹⁵N-enrichment or ¹⁵N-natural abundance have assumed a dominant role in quantifying N cycle processes involving BFN. However, it is only through the direct ¹⁵N₂ approach that biological N₂ fixation can be traced through the various components of the soil-plant system. Technological advances in the automated control of the chamber environment have made the ¹⁵N₂ technique more attractive to long-term studies. Thus the need to enclose plants in a chamber and maintain conditions conducive to plant growth should no longer be seen as a major obstacle to the use of ¹⁵N₂. The way is now open to evaluate the efficacy of indirect methods used to estimate the contribution of BFN to the N economies of crop and pasture systems, and the dynamics of BFN in agroecosystems. In addition, new applications of ¹⁵N₂ such as stable isotope probing are emerging, which have the potential to characterize non-cultivated diazotrophs in a range of environments. The role of biological N₂ fixation in the formation of reactive N in the environment and its relationship with the emission of the greenhouse gas N₂O requires further investigation.

Introduction

¹⁵N₂, or to be more precise ¹⁵N₂ reduction, has played a key role in studies of biological N₂ fixation over the past 75 years, since publication of the seminal paper of Burris and Miller (1941). Several general reviews have been written about the measurement of N₂ fixation using ¹⁵N techniques, including ¹⁵N₂ (Bergersen, 1980, Focht and Poth, 1987, Weaver and Danso, 1994, Zehr and Montoya, 2007). However, a contemporary overview of the subject is lacking, particularly the role of ¹⁵N₂ as the only direct ¹⁵N method. The major drawback of using ¹⁵N₂ is the high cost of the isotope which necessitates exposure of the plant to the substrate in a leak-free closed chamber, thus precluding the use of more realistic experimental set-ups such as open-top or flow-through chambers.

Millbank and Olsen (1981) and Warembourg (1993) provided descriptions of (i) systems to prepare, purify and store ¹⁵N₂ (ii) the types of enclosures used for biological materials (iii) methods of environmental control of enclosures and (iv) operating procedures. Many other enclosure systems that were not reviewed by Millbank and Olsen (1981) or Warembourg (1993) have been described in the literature. Consequently we will provide an overview of the distinctive features of diverse systems used with vascular plants, including legumes and non-legumes.

In addition, we will explore the usage of ¹⁵N₂ in fundamental studies of N₂ fixation, including confirmation of putative N₂ fixation in various biological systems, the calibration of assays to estimate the rate of biological N₂ fixation, the identification of the biochemical products of N₂ fixation and assimilation pathways within the plant, ¹⁵N₂ stable isotope probing to identify uncultured diazotrophs in the biosphere and the role of ¹⁵N₂ in quantifying N₂ fixation and the dynamics of BFN within various components of terrestrial and fresh water ecosystems. We will discuss the practical problems encountered when employing ¹⁵N₂ as a quantitative tracer, and highlight the problems associated with alternative indirect ¹⁵N methods. The focus of the review is terrestrial ecosystems, particularly agroecosystems, which also include flooded rice, but we do not cover the marine environment.

Section snippets

Cultures, excised nodules/nodulated roots, soil, and non-vascular plants

Burris and Miller (1941) were the first to report N₂ fixation by a culture of Azotobacter vinelandii by exposure to ¹⁵N₂, while Burris et al. (1943) exposed excised nodules and nodulated roots of pea to ¹⁵N₂ to demonstrate legume-Rhizobium symbiotic N₂ fixation. Subsequent studies with ¹⁵N₂ confirmed biological N₂ fixation with lichens and the liverwort Blasia (Bond and Scott, 1955), and free-living heterotrophs in soil (Delwiche and Wijler, 1956). Later work with ¹⁵N₂ focused on N₂ fixation in

Systems used to expose biological materials to ¹⁵N₂

Various types of chambers have been constructed to enclose the roots or the roots + tops of the plant, with considerable variation in chamber volume (Table 1). Warembourg (1993) outlined the factors to be considered when deciding on chamber volume. Often relatively small volumes (<3 l) were used to reduce the cost of the isotope (e.g. Bond, 1955, De-Polli et al., 1977, Ruschel et al., 1979, Frey and Schüepp, 1992, Bremer et al., 1995), but some chambers were much larger varying from 60 l (

Identification of the biochemical products of biological N₂ fixation and assimilation pathways

Various physiological studies since the mid 1940's have supplied ¹⁵N₂ to free-living N₂-fixing micro-organisms (Burris and Wilson, 1946, Zelitch et al., 1951), detached alder nodules (Alnus glutinosa; Leaf et al., 1958), detached bog-myrtle nodules (Myrica gale; Leaf et al., 1959), detached legume nodules (Aprison et al., 1954, Bergersen, 1965, Kennedy, 1966a, Kennedy, 1966b), isolated bacteroids (the N₂-fixing forms of rhizobia present in nodule infected cells) from legume nodules (Bergersen

Calibration of assay procedures using ¹⁵N₂

Numerous studies that were designed to calibrate the acetylene reduction assay were conducted with ¹⁵N₂ using microbial cultures, excised nodules, excised nodulated roots, plant litter, stem segments, intact root systems and whole plants (Table 3). Calibration involved the simultaneous determination of the ratio of C₂H₂ reduced to N₂ fixed. The ratios obtained were extremely variable being either higher or lower than the theoretical 3:1 (Table 3). Many factors were responsible for this

Stable isotope probing (SIP) with ¹⁵N₂

Despite the vital importance of biological N₂ fixation in maintaining terrestrial ecosystem sustainability, the taxonomic identity of the microorganisms involved has usually been confined to a small fraction of the microbiota that can be isolated and cultivated. The recent development in the coupling of molecular biological methods with SIP in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment (Radajewski et al.,

Endophytic diazotrophs

¹⁵N₂ has been used worldwide to identify endophytic biological N₂ fixation in a range of cereals and tropical grasses. Chalk (2016) reviewed 22 published papers in which the ¹⁵N₂ technique confirmed putative N₂ fixation. Because a control or reference treatment is not required, N₂ fixation can be measured either under natural (undisturbed) or imposed (e.g. inoculated) treatments. Exposure times were generally of short duration, usually a few days but seldom exceeding 1–2 weeks (Chalk, 2016).

Studies with rice

The distribution of ¹⁵N₂ fixed between above- and below-ground components of paddy rice was determined by short- and long-term exposure to ¹⁵N₂ at different growth stages (Table 5). The highest proportion of N₂ fixed for short-term exposure was in the soil (>75%), with a greater proportion in tops than in roots, irrespective of growth stage, exposure time or whether roots or tops + roots were exposed to ¹⁵N₂ (Table 5).

For a long-term exposure to ¹⁵N₂ (10 weeks) a different distribution emerged

P_atm estimated by indirect methods that use a reference plant

Indirect ¹⁵N-based methodologies using either E or NA approaches to estimate P_atm by N₂-fixing plant associations require a non-N₂-fixing reference plant treatment to act as a surrogate to estimate the proportion of labelled to unlabelled soil N obtained by the N₂ fixer (Unkovich et al., 2008, Chalk et al., 2016). The E method is based on the addition of a ¹⁵N-enriched material to the soil, but no addition is required with the NA method. The E method results in a marked temporal and spatial

Conclusions

¹⁵N₂ has been used on many occasions since 1941 to examine the assimilatory pathways associated with biological N₂ fixation, and to confirm putative N₂ fixation by a range of biological materials. However, because of technical problems associated with the use of enclosures and the high cost of ¹⁵N₂, the direct method has seldom been used to obtain quantitative estimates of N₂ fixed over a plant's life cycle. The relatively small chamber volume has generally restricted the scale of experiments

Acknowledgements

The senior author thanks the Chinese Academy of Sciences (CAS) for a Visiting Fellowship under the CAS President's International Fellowship Initiative for 2016, Grant No. 2016VMB029.

References (117)

S.L. Addison et al.
Stable isotope probing: technical considerations when resolving ¹⁵N-labeled RNA in gradients
Journal of Microbiological Methods
(2010)
M.H. Aprison et al.
Nitrogen fixation by excised soy bean root nodules
Journal of Biological Chemistry
(1954)
M. Becker et al.
Growth and N₂-fixation of two stem-nodulating legumes and their effect as green manure on lowland rice
Soil Biology & Biochemistry
(1990)
Q. Bei et al.
Heterotrophic and phototrophic ¹⁵N₂ fixation and distribution of fixed ¹⁵N in a flooded rice–soil system
Soil Biology & Biochemistry
(2013)
D.H. Buckley et al.
¹⁵N₂–DNA–stable isotope probing of diazotrophic methanotrophs in soil
Soil Biology & Biochemistry
(2008)
R.H. Burris
Nitrogen fixation – assay methods and techniques
Methods in Enzymology
(1972)
R.H. Burris et al.
Detection of nitrogen fixation with isotopic nitrogen
The Journal of Biological Chemistry
(1943)
R.H. Burris et al.
Comparison of the metabolism of ammonia and molecular nitrogen in azotobacter
Journal of Biological Chemistry
(1946)
P.M. Chalk et al.
Estimation of legume symbiotic dependence: an evaluation of techniques based on ¹⁵N dilution
Soil Biology & Biochemistry
(1999)
P.M. Chalk et al.
Methodologies for estimating nitrogen transfer between legumes and companion species in agro-ecosystems: a review of ¹⁵N-enriched techniques
Soil Biology & Biochemistry
(2014)

O.T. Denmead et al.

A closed ammonia cycle within a plant canopy

Soil Biology & Biochemistry

(1976)

H. De-Polli et al.

Confirmation of nitrogen fixation in two tropical grasses by ¹⁵N₂ incorporation

Soil Biology & Biochemistry

(1977)

R. Diocares et al.

Producing and dispensing small quantities of ¹⁵N₂ gas at atmospheric pressure

Analytical Biochemistry

(2006)

K.E. Giller

Use and abuse of the acetylene reduction assay for measurement of “associative” nitrogen fixation

Soil Biology & Biochemistry

(1987)

K.E. Giller et al.

A method for measuring the transfer of fixed nitrogen from free-living bacteria to higher plants using ¹⁵N₂

Journal of Microbiological Methods

(1984)

A.R. Harker et al.

Transfer of ¹⁵N₂ to closed systems in the field

Soil Biology & Biochemistry

(1981)

I.R. Kennedy

Primary products of symbiotic nitrogen fixation I. Short-term exposures of serradella nodules to ¹⁵N₂

Biochimica et Biophysica Acta

(1966)

I.R. Kennedy

Primary products of symbiotic nitrogen fixation II. Pulse-labelling of serradella nodules with ¹⁵N₂

Biochimica et Biophysica Acta

(1966)

A. Keuter et al.

Asymbiotic biological nitrogen fixation in a temperate grassland as affected by management practices

Soil Biology & Biochemistry

(2014)

R.M. Mohr et al.

Fate of symbiotically-fixed ¹⁵N₂ as influenced by method of alfalfa termination

Soil Biology & Biochemistry

(1998)

M.B. Peoples et al.

Can differences in ¹⁵N natural abundance be used to quantify the transfer of nitrogen from legumes to non-legume plant species?

Soil Biology & Biochemistry

(2015)

S. Radajewski et al.

Stable isotope probing of nucleic acids: a window to the function of uncultured microorganisms

Current Opinion in Biotechnology

(2003)

J.P. Roskoski

Comparative C₂H₂ reduction and N₂ fixation in deciduous wood litter

Soil Biology & Biochemistry

(1981)

S.L. Addison et al.

Identifying diazotrophs by incorporation of nitrogen from ¹⁵N₂ into RNA

Applied Microbiology and Biotechnology

(2010)

S. Akao

Nitrogen fixation and metabolism in soybean plants

JARQ

(1991)

D. Allaway et al.

Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids

Molecular Microbiology

(2000)

C.A. Atkins et al.

Amino acid transport and metabolism in relation to the nitrogen economy of a legume leaf

Plant Physiology

(1983)

C.A. Atkins et al.

Metabolism and translocation of allantoin in ureide-producing grain legumes

Plant Physiology

(1982)

C.A. Atkins et al.

Assimilation of fixed-N in a ureide-forming symbiosis

D. Barraclough

The use of mean pool abundances to interpret ¹⁵N tracer experiments. I. Theory

Plant and Soil

(1991)

K. Basilier

Fixation and uptake of nitrogen by Sphagnum blue-green algal associations

Oikos

(1980)

F.J. Bergersen

Ammonia – an early stable product of nitrogen fixation by soybean root nodules

Australian Journal of Biological Sciences

(1965)

F.J. Bergersen

Measurement of nitrogen fixation by direct means

F.J. Bergersen et al.

Nitrogen fixation in the coralloid roots of Macrozamia communis L. Johnson

Australian Journal of Biological Sciences

(1965)

F.J. Bergersen et al.

Bacteroids from soybean root nodules : respiration and N₂-fixation in flow-chamber reactions with oxyleghaemoglobin

Proceedings of the Royal Society of London B

(1990)

G. Bond

An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus, Myrica, and Hippophaë

Journal of Experimental Botany

(1955)

G. Bond et al.

An examination of some symbiotic systems for fixation of nitrogen

Annals of Botany

(1955)

E. Bremer et al.

Evidence against associative N₂ fixation as a significant N source in long-term wheat plots

Plant and Soil

(1995)

R. Brouzes et al.

The effect of organic amendment, water content, and oxygen on the incorporation of ¹⁵N₂ by some agricultural and forest soils

Canadian Journal of Microbiology

(1969)

D.H. Buckley et al.

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

Applied and Environmental Microbiology

(2007)

R.H. Burris et al.

Application of N¹⁵ to the study of biological nitrogen fixation

Science

(1941)

M.S. Carter et al.

Biologically-fixed N₂ as a source for N₂O production in a grass–clover mixture, measured by ¹⁵N₂

Nutrient Cycling in Agroecosystems

(2006)

P.M. Chalk

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

Plant and Soil

(1991)

P.M. Chalk

Dynamics of biologically-fixed N in legume-cereal rotations: a review

Crop & Pasture Science

(1998)

P.M. Chalk

The strategic role of ¹⁵N in quantifying the contribution of endophytic N₂ fixation to the N nutrition of non-legumes

Symbiosis

(2016)

P.M. Chalk et al.

Do techniques based on ¹⁵N enrichment and ¹⁵N natural abundance give consistent estimates of the symbiotic dependence of N₂-fixing plants?

Plant and Soil

(2016)

P.M. Chalk et al.

A yield-independent, ¹⁵N-isotope dilution method to estimate legume symbiotic dependence without a non-N₂-fixing reference plant

Biology and Fertility of Soils

(1996)

P.C. Chang et al.

Non-symbiotic nitrogen fixation in some Quebec soils

Canadian Journal of Microbiology

(1965)

C.C. Cleveland et al.

Global patterns of terrestrial biological nitrogen (N₂) fixation in natural ecosystems

Global Biogeochemical Cycles

(1999)

R. Dabundo et al.

The contamination of commercial ¹⁵N₂ gas stocks with ¹⁵N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements

PloS one

(2014)

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Review Paper15N2 as a tracer of biological N2 fixation: A 75-year retrospective

Highlights

Abstract

Introduction

Section snippets

Cultures, excised nodules/nodulated roots, soil, and non-vascular plants

Systems used to expose biological materials to 15N2

Identification of the biochemical products of biological N2 fixation and assimilation pathways

Calibration of assay procedures using 15N2

Stable isotope probing (SIP) with 15N2

Endophytic diazotrophs

Studies with rice

Patm estimated by indirect methods that use a reference plant

Conclusions

Acknowledgements

Journal of Microbiological Methods

Journal of Biological Chemistry

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Methods in Enzymology

The Journal of Biological Chemistry

Journal of Biological Chemistry

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Analytical Biochemistry

Soil Biology & Biochemistry

Journal of Microbiological Methods

Soil Biology & Biochemistry

Biochimica et Biophysica Acta

Biochimica et Biophysica Acta

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Soil Biology & Biochemistry

Current Opinion in Biotechnology

Soil Biology & Biochemistry

Identifying diazotrophs by incorporation of nitrogen from 15N2 into RNA

Applied Microbiology and Biotechnology

Nitrogen fixation and metabolism in soybean plants

JARQ

Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids

Molecular Microbiology

Amino acid transport and metabolism in relation to the nitrogen economy of a legume leaf

Plant Physiology

Metabolism and translocation of allantoin in ureide-producing grain legumes

Plant Physiology

Assimilation of fixed-N in a ureide-forming symbiosis

The use of mean pool abundances to interpret 15N tracer experiments. I. Theory

Plant and Soil

Fixation and uptake of nitrogen by Sphagnum blue-green algal associations

Oikos

Ammonia – an early stable product of nitrogen fixation by soybean root nodules

Australian Journal of Biological Sciences

Measurement of nitrogen fixation by direct means

Nitrogen fixation in the coralloid roots of Macrozamia communis L. Johnson

Australian Journal of Biological Sciences

Bacteroids from soybean root nodules : respiration and N2-fixation in flow-chamber reactions with oxyleghaemoglobin

Proceedings of the Royal Society of London B

An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus, Myrica, and Hippophaë

Journal of Experimental Botany

An examination of some symbiotic systems for fixation of nitrogen

Annals of Botany

Evidence against associative N2 fixation as a significant N source in long-term wheat plots

Plant and Soil

The effect of organic amendment, water content, and oxygen on the incorporation of 15N2 by some agricultural and forest soils

Canadian Journal of Microbiology

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

Applied and Environmental Microbiology

Application of N15 to the study of biological nitrogen fixation

Science

Biologically-fixed N2 as a source for N2O production in a grass–clover mixture, measured by 15N2

Nutrient Cycling in Agroecosystems

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

Plant and Soil

Dynamics of biologically-fixed N in legume-cereal rotations: a review

Crop & Pasture Science

The strategic role of 15N in quantifying the contribution of endophytic N2 fixation to the N nutrition of non-legumes

Symbiosis

Review Paper
¹⁵N₂ as a tracer of biological N₂ fixation: A 75-year retrospective

Systems used to expose biological materials to ¹⁵N₂

Identification of the biochemical products of biological N₂ fixation and assimilation pathways

Calibration of assay procedures using ¹⁵N₂

Stable isotope probing (SIP) with ¹⁵N₂

P_atm estimated by indirect methods that use a reference plant

Identifying diazotrophs by incorporation of nitrogen from ¹⁵N₂ into RNA

The use of mean pool abundances to interpret ¹⁵N tracer experiments. I. Theory

Bacteroids from soybean root nodules : respiration and N₂-fixation in flow-chamber reactions with oxyleghaemoglobin

Evidence against associative N₂ fixation as a significant N source in long-term wheat plots

The effect of organic amendment, water content, and oxygen on the incorporation of ¹⁵N₂ by some agricultural and forest soils

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

Application of N¹⁵ to the study of biological nitrogen fixation

Biologically-fixed N₂ as a source for N₂O production in a grass–clover mixture, measured by ¹⁵N₂

The strategic role of ¹⁵N in quantifying the contribution of endophytic N₂ fixation to the N nutrition of non-legumes

Do techniques based on ¹⁵N enrichment and ¹⁵N natural abundance give consistent estimates of the symbiotic dependence of N₂-fixing plants?

A yield-independent, ¹⁵N-isotope dilution method to estimate legume symbiotic dependence without a non-N₂-fixing reference plant

Global patterns of terrestrial biological nitrogen (N₂) fixation in natural ecosystems

The contamination of commercial ¹⁵N₂ gas stocks with ¹⁵N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements