2.2.11 Biological nitrogen fixation

Authors: Van Langenhove L1, Vicca S1

Reviewers: Ribbons R2


Measurement unit: N ha-1 y-1; Measurement scale: plot; Equipment costs: none; Running costs: €€€€€; Installation effort: medium; Maintenance effort: medium; Knowledge need: medium; Measurement mode: manual

Biological nitrogen fixation (BNF) is the largest natural source of exogenous nitrogen (N) in unmanaged ecosystems (Vitousek et al., 2013) and also the primary baseline against which anthropogenic changes to the N cycle are measured (Vitousek et al., 1997). BNF arises from both symbiotic associations in the form of root nodules between bacteria and plants as well as free-living microorganisms (e.g. in leaf litter and soil), called diazotrophs (Reed et al., 2011). Broad-scale estimates of BNF in natural temperate ecosystems, especially in late-successional temperate grasslands and forests, are consistently low, while the highest rates of naturally occurring BNF presumably occur in the evergreen lowland tropical rainforest (Cleveland et al., 1999; Vitousek et al., 2013). Low estimates of BNF in temperate grasslands and forests are mainly due to a scarcity of symbiotically fixing plant species in these ecosystems. Recently, however, Moyes et al., (2016) found diazotrophs living inside pine tree needles, providing an understudied source of N to temperate and boreal pine forests. In agricultural systems, crop legumes that symbiotically fix atmospheric N are commonly used as a renewable source of N in agriculture and their N-fixing capacity depends on microbial, soil, and environmental variables (Peoples et al., 1995). Tropical rainforests contain a high abundance and diversity of putative symbiotically fixing plant species and studies have shown that tree nodulation, and by extension symbiotic N fixation, is influenced by forest maturity and disturbance (Barron et al., 2011; Bauters et al., 2016). Barron et al. (2011) concluded that the observed pattern is evidence of the suspected inverse relationship between soil nitrate levels and nodulation, as consistent with feedback control by local N availability. There appears to be a relationship between phosphorus (P), molybdenum (Mo), and free-living BNF (Wurzburger et al., 2012; Reed et al., 2013), where BNF is constrained by P, Mo, or both. The interplay between these two elements in the organic soil layer has been put forward as an important determinant of free-living BNF rates in various neotropical forests (Wurzburger et al., 2012; Reed et al., 2013), temperate grasslands, temperate forests (Jean et al., 2013), and boreal forests (Rousk et al., 2017a). The determination of ecosystem-wide rates and controls of BNF is crucial to placing anthropogenic changes to the N cycle in context (Vitousek et al., 2013). The large-scale effects of a changing climate on BNF remain unknown, as there are only a few studies that have measured BNF after temperature and/or moisture manipulation. However, a forest inventory study suggests that N-fixing tree abundance will increase with climate warming (Liao et al., 2017), and a study on boreal mosses reported N fixation increases in response to higher temperatures (Rousk et al., 2017b) What and how to measure?

Gold standard

The most accurate determination of N fixed over a given time period is a direct measurement through the use of isotopically labelled N: 15N2 gas, called the 15N tracer method. First a sample is taken in the field. This can be a soil sample (preferably topsoil as N2 fixation rates decline rapidly with depth), vegetation sample (fresh leaf, fallen litter) or roots with nodules. Typically, samples are small (+- 25 g for soil or a few leaves or roots) and will immediately be placed in the incubation chamber. On the day of sampling, the chamber is enriched with 15N2 and samples are allowed to fixate during a predetermined time period. Initiation of the incubation should be done as fast as possible because most research questions concern in vivo rates of N2 fixation and thus changes in microbial activity should be avoided. After incubation, the sample is dried, ground, and analysed with a mass spectrometer to determine the ratio between 14N and 15N on a weight basis (Furnkranz et al., 2008). This ratio is then compared to the 14N / 15N ratio of a sample blank, which was incubated in regular air in parallel, and used to calculate the amount of N that was fixed during the incubation period. Once the sample is dried, it can be stored for several months as long as there is no contact with other 15N sources. The main advantages of this method are that it is a direct measurement of N fixation and it is the only technique that unequivocally proves N fixation. For chamber enrichment, the highest quality of 15N should be used, so at least 98 % 15N. Unfortunately, this gas is very expensive, as is the required isotope-ratio mass spectrometer that measures the small increases in the 15N / 14N ratio, making the method unsuitable for the incubation of large volumes (Unkovich et al., 2008).


Bronze method

The enzyme nitrogenase, responsible for the reduction of N2 into ammonia (NH3) in diazotrophs, is also capable of reducing acetylene (C2H2) into ethylene (C2H4) (Hardy et al., 1968). Thus, acetylene can be used as an alternative substrate to N2, in a method called the Acetylene Reduction Assay (ARA; Hardy et al., 1968). This method is suitable for similar sample types as the 15N tracer method. On the day of sampling, samples are placed in an airtight chamber or cuvette and exposed to a C2H2 enriched atmosphere (usually 10 % C2H2 in air) for the duration of the incubation. Incubation in an C2H2 enriched atmosphere can be done immediately in the field or after transportation in the lab. The incubation time is seen as a trade-off between ethylene accumulation and isolation within a closed-off environment. Typically, samples with high fixation rates, such as root nodules, are incubated for anywhere between 30 min to a couple of hours (e.g. Menge & Hedin, 2009), while samples known to possess lower fixation rates, such as tropical topsoil or boreal bryophytes, are incubated for 18–24 h (e.g. Černá et al., 2009; Rousk & Michelsen, 2016). The rate of C2H4 accumulation in gas samples collected over a period of time is measured by a gas chromatograph (Vessey, 1994). Gas samples are recommended to be analysed as quickly as possible to avoid problems with leaking sampling vials. Given that acetylene is cheap and easy to make (if so desired) in the lab, and the technique is not very labour intensive, many measurements can be undertaken on a daily basis. The sensitivity of this method to detect nitrogenase activity is unparalleled, yet it does not directly measure N fixation and relies on the conversion of produced C2H2 into N fixed for quantification of N2 fixation. While there is theoretical and empirical support for a conversion ratio of C2H4 produced to N2 fixed of 3–4 to 1 (Nohrstedt et al., 1983), C2H2 reduction assays should ideally be calibrated by means of 15N2. Lastly, there is also evidence for a decline in nitrogenase activity after exposure to C2H2 in some legume species (Hunt & Layzell, 1993), casting even more doubt upon the quantitative aspects of the assay.


Installation, field operation, maintenance, interpretation

The operation in field and lab is similar for free-living and symbiotic BNF and besides the sensitive measuring equipment, such as the gas chromatograph and mass spectrometer, no maintenance is required.


Symbiotic BNF

Depending on your research question, root nodules are excavated and can be (i) incubated either detached from roots or (ii) the root system can be incubated entirely (Peoples et al., 2009). The first will yield a fixation rate per gram of nodule (g N fixed g-1 h-1) and will need a complementary measurement of the nodule density (g plant-1) and N concentration per plant along with knowledge of the amount of plants per plot to be able to upscale the results. The second is not applicable for trees, but in the case of small crop legumes it gives a good estimate of N fixation at the plot level (kg N ha-1 y-1), provided the biomass of crop legume roots (kg ha-1) is known. The estimation of plant dry matter, N concentration, and nodule density represent a considerable source of error in field estimates.


Free-living BNF

A sample from the substrate on which BNF is to be measured is incubated. Since free-living BNF can be very patchy and so called “hotspots” (Alexander & Schell, 1973; Pérez et al., 2008; Reed et al., 2010) with higher fixation rates than the surrounding areas have been observed, it is paramount to have a sampling strategy that covers the desired plot well. The resulting fixation rate will typically be expressed in N fixed per gram of substrate per time unit (mg N g-1 h-1) in the case of soil or litter or expressed in N fixed per area per time unit (mg N cm-2 h-1) in the case of tree phyllosphere (Furnkranz et al., 2008). Usually, this value is up-scaled and reported for the plot level (kg N ha-1 y-1) through knowledge of soil density (kg m-3) and litter density (kg m-2). Upscaling the phyllosphere fixation rates is harder, since knowledge about the leaf surface per tree (m² tree-1) and trees per plot (tree ha-1) is required. Plant trait databases, such as TRY (Kattge et al., 2011), can help find this information.

It is important to keep in mind that fixation hotspots cause large errors in up-scaling and the free-living fixation rate often displays strong seasonal variation (Reed et al., 2007; Pérez et al., 2008), making several samplings per year to capture all seasons a necessity.


Where to start

Furnkranz et al. (2008), Hardy et al. (1968), Herridge et al. (2008), Reed et al. (2011), Unkovich et al. (2008) Special cases, emerging issues, and challenges

One of the main challenges in nitrogen fixation studies is quantifying N2 fixation in environments where fixation is expected to be low or sporadic, such as in soils. Using the acetylene assay in these environments may not produce detectable ethylene concentrations as production rates are low and the incubation time short. Prolonging the incubation time may help solve this problem. However, it may also introduce possibly unwanted side effects, such as the initiation of the anaerobic metabolism or decrease of the moisture content, due to prolonged isolation of the sample from the environment (O’Toole & Knowles, 1973). In these instances it may be wise to replace the acetylene assay with the 15N tracer technique, as it is more sensitive, although this does not always result in detectable fixation rates. In soils in particular, background N pools (primarily stemming from organic matter) may be large and the increase in 15N:14N ratio unnoticeable compared to this background pool (Angel et al., 2018).

An emerging technique of bypassing the diluting effects of a large background N pool in soils is to measure the 15N enrichment in specific microbial biomolecules, such as DNA, RNA, and proteins (Angel at al., 2018). Increases in 15N within any of these molecules would indicate N2 fixation and, provided an increase in DNA is detected, growth. As it stands, the main challenges for this technique will be i) application, as it is not generally applicable across diverse microbial species, ii) up-scaling, as the technique requires a great amount of time to perform, limiting the amount of samples that can be processed, and iii) costs, as 15N2 tracer gas is expensive and access to an isotope ratio mass spectrometer and a microbial laboratory is needed for all manipulations involving DNA, RNA, and proteins. In spite of its drawbacks, the application of this technique has the potential of studying free-living diazotrophs in environments where it was thus far nearly impossible. References

Theory, significance, and large datasets

We recommend Reed et al. (2011) and Herridge et al. (2008) for information on biological N fixation in natural ecosystems and agricultural systems, respectively.


More on methods and existing protocols

For more detailed information on different protocols to measure N fixation we recommend Unkovich et al. (2008). Furnkranz et al. (2008) describes how to measure N fixation using the 15N isotopic method and the report by Hardy et al. (1968) provides detailed benefits and drawbacks of the acetylene reduction assay.


All references

Alexander, V., & Schell, D. M. (1973). Seasonal and spatial variation of nitrogen fixation in the Barrow, Alaska, Tundra. Arctic and Alpine Research, 5(2), 77-88.

Angel, R., Panholzl, C., Gabriel, R., Herbold, C., Wanek, W., Richter, A., … Woebken, D. (2018). Application of stable-isotope labelling techniques for the detection of active diazotrophs. Environmental Microbiology, 20(1), 44-61

Barron, A. R., Purves, D. W., & Hedin, L. O. (2011). Facultative nitrogen fixation by canopy legumes in a lowland tropical forest. Oecologia, 165(2), 511-520.

Bauters, M., Mapenzi, N., Kearsley, E., Vanlauwe, B., & Boeckx, P. (2016). Facultative nitrogen fixation by legumes in the central Congo basin is downregulated during late successional stages. Biotropica, 48(3), 281-284.

Černá, B., Rejmánková, E., Snyder, J. M., & Šantrůčková, H. (2009). Heterotrophic nitrogen fixation in oligotrophic tropical marshes: changes after phosphorus addition. Hydrobiologia, 627(1), 55-65.

Cleveland, C. C., Townsend, A. R., Schimel, D. S., Fisher, H., Howarth, R. W., Hedin, L. O., … Wasson, M. F. (1999). Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biogeochemical Cycles, 13(2), 623-645.

Furnkranz, M., Wanek, W., Richter, A., Abell, G., Rasche, F., & Sessitsch, A. (2008). Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica. ISME Journal, 2(5), 561-570.

Jean, M. E., Phalyvong, K., Forest-Drolet, J., & Bellenger, J. P. (2013). Molybdenum and phosphorus limitation of asymbiotic nitrogen fixation in forests of Eastern Canada: Influence of vegetative cover and seasonal variability. Soil Biology and Biochemistry, 67, 140-146.

Hardy, R. W., Holsten, R. D., Jackson, E. K., & Burns, R. C. (1968). The acetylene-ethylene assay for N(2) fixation: laboratory and field evaluation. Plant Physiology, 43(8), 1185-1207.

Herridge, D. F., Peoples, M. B., & Boddey, R. M. (2008). Global inputs of biological nitrogen fixation in agricultural systems. Plant and Soil, 311(1-2), 1-18.

Hunt, S., & Layzell, B. D. (1993). Gas exchange of legume nodules and the regulation of nitrogenase activity. Annual Review of Plant Physiology and Plant Molecular Biology, 44(1), 483-511.

Kattge, J., Díaz, S., Lavorel, S., Prentice, I. C., Leadley, P., Bönisch, G., … Wirth, C. (2011). TRY – a global database of plant traits. Global Change Biology, 17(9), 2905-2935.

Liao, W., Menge, D. N. L., Lichstein, J. W., & Ángeles‐Pérez, G. (2017). Global climate change will increase the abundance of symbiotic nitrogen-fixing trees in much of North America. Global Change Biology, 23(11), 4777-4787.

Menge, D. N., & Hedin, L. O. (2009). Nitrogen fixation in different biogeochemical niches along a 120,000-year chronosequence in New Zealand. Ecology, 90(8), 2190-2201.

Moyes, A. B., Kueppers, L. M., Pett-Ridge, J., Carper, D. L., Vandehey, N., O’Neil, J., & Frank, A. C. (2016). Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer. New Phytologist, 210(2), 657-668.

Nohrstedt, H.-Ö. (1983). Conversion factor between acetylene reduction and nitrogen fixation in soil: Effect of water content and nitrogenase activity. Soil Biology and Biochemistry, 15(3), 275-279

O’Toole, P., & Knowles, R. (1973). Oxygen inhibition of acetylene reduction (nitrogen fixation) in soil: Effect of glucose and oxygen concentrations. Soil Biology and Biochemistry, 5(6), 783-788.

Peoples, M. B., Ladha, J. K., & Herridge, D. F. (1995). Enhancing legume N2 fixation through plant and soil management. Plant and Soil, 174(1), 83-101.

Peoples, M. B., Unkovich, M. J., & Herridge, D. F. (2009). Measuring symbiotic nitrogen fixation by legumes. In D. W. Emerich & H. Krishnan (Eds.), Nitrogen Fixation in Crop Production. (Agronomy Monograph Vol. 52; pp. 125-170).

Pérez, S., Pérez, C., Carmona, M., Farina, J., & Armesto, J. (2008). Effects of labile phosphorus and carbon on non-symbiotic N2 fixation in logged and unlogged evergreen forests in Chiloé Island, Chile. Revista Chilena de Historia Natural, 81, 267-278.

Reed, S. C., Cleveland, C. C., & Townsend, A. R. (2007). Controls over leaf litter and soil nitrogen fixation in two lowland tropical rain forests. Biotropica, 39(5), 585-592.

Reed, S. C., Townsend, A. R., Cleveland, C. C., & Nemergut, D. R. (2010). Microbial community shifts influence patterns in tropical forest nitrogen fixation. Oecologia, 164(2), 521-531.

Reed, S. C., Cleveland, C. C., & Townsend, A. R. (2011). Functional ecology of free-living nitrogen fixation: a contemporary perspective. Annual Review of Ecology, Evolution, and Systematics, 42, 489-512.

Reed, S. C., Cleveland, C. C., & Townsend, A. R. (2013). Relationships among phosphorus, molybdenum and free-living nitrogen fixation in tropical rain forests: results from observational and experimental analyses. Biogeochemistry, 114(1-3), 135-147.

Rousk, K., & Michelsen, A. (2016). The sensitivity of moss-associated nitrogen fixation towards repeated nitrogen input. PLoS One, 11(1), e0146655.

Rousk, K., Degboe, J., Michelsen, A., Bradley, R., & Bellenger, J. P. (2017a). Molybdenum and phosphorus limitation of moss-associated nitrogen fixation in boreal ecosystems. New Phytologist, 214(1), 97-107.

Rousk, K., Pedersen, P. A., Dyrnum, K., & Michelsen, A. (2017b). The interactive effects of temperature and moisture on nitrogen fixation in two temperate-arctic mosses. Theoretical and Experimental Plant Physiology, 29(1), 25-36.

Unkovich, M., Herridge, D. F., Peoples, M. B., Boddey, R. M., Cadisch, G., Giller, K. E., … Chalk, P. (2008). Measuring Plant-Associated Nitrogen Fixation in Agricultural Systems. Canberra: Australian Centre for International Agricultural Research.

Vessey, J. K. (1994). Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay. Plant and Soil, 158(2), 151-162.

Vitousek, P. M., Aber, J. D., Howarth, R. W., Likens, G. E., Matson, P. A., Schindler, D. W., … Tilman, D. G. (1997). Human alteration of the global nitrogen cycle: sources and consequences. Ecological Applications, 7(3), 737-750.

Vitousek, P. M., Menge, D. N., Reed, S. C., & Cleveland, C. C. (2013). Biological nitrogen fixation: rates, patterns and ecological controls in terrestrial ecosystems. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 368(1621), 20130119.

Wurzburger, N., Bellenger, J. P., Kraepiel, A. M., & Hedin, L. O. (2012). Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests. PLoS One, 7(3), e33710.



Authors: Van Langenhove L1, Vicca S1

Reviewers: Ribbons R2



1 Centre of Excellence PLECO (Plants and Ecosystems), Biology Department, University of Antwerp, Wilrijk, Belgium

2 Biology and Geology Departments, Lawrence University, Appleton, USA