Skip to main content
SpringerLink
Log in

Assessment of biological nitrogen fixation

  • Chapter
Nitrogen Economy in Tropical Soils

Part of the book series: Developments in Plant and Soil Sciences ((DPSS,volume 69))

  • 432 Accesses

Abstract

The four commonly used methods for measuring biological nitrogen fixation (BNF) in plants are: the total nitrogen difference (TND) method, acetylene reduction assay (ARA) technique, xylem-solute (or ureide production) method and the use of 15N labelled compounds.

The TND method relies on a control non-N2-fixing plant to estimate the amount of N absorbed by the fixing plant from soil. It is one of the simplest and least expensive methods, but works best under low soil N conditions. The ARA technique measures the rate of acetylene conversion to ethylene by the N2-fixing enzyme, nitrogenase. The ethylene produced can then be converted into N2 fixed, using a conversion ratio, originally recommended as 3. Although the method is inexpensive and highly sensitive, its major disadvantages are, the short-term nature of the assays, the doubtful validity of always using a conversion ratio of 3 and the auto-inhibition of acetylene conversion to ethylene. The ARA technique is therefore not a method of choice for measuring BNF.

The xylem-solute technique can be used to measure BNF for those species that produce significant quantities of ureide as product of BNF. Although simple and relatively inexpensive, it is an instantaneous assay and also needs to be calibrated against a known method. The most serious limitation is, that only a small proportion of N2-fixing plants examined are ureide exporters, and the method is therefore not widely applicable.

The 15N methods, classified into the isotope dilution and A-value methods, appear to be the most accurate, but also the most expensive. They involve labelling soil with 15N fertilizer and using a non-N2-fixing reference plant to measure the 15N/14N ratio in the soil. The 15N isotope dilution approach is both operationally and mathematically simpler than the A-value approach. To limit potential errors in the selection of reference crops, it is recommended to use 15N labelled compounds or soil labelling methods that result in the slow release of 15N or the slow decline of 15N/14N ratio in the soil. Additionally, the use of several reference plants rather than a single one can improve the accuracy of the results.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 259.00
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 329.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Similar content being viewed by others

References

  1. Atkins CA, Pate JS, Griffiths GJ and White ST (1980) Economy of carbon and nitrogen in nodulated and non-nodulated NO3-grown cowpea [Vigna unguiculata (L.) Walp.]. Plant Physiol. 66: 978–903

    Article  CAS  Google Scholar 

  2. Attewell J and Bliss F A (1985) Host plant characteristics of common bean line selected using indirect measures of N2 fixation. In: Evans HJ, Bottomley PJ and Newton WE (eds) Nitrogen Fixation Progress, pp 3–9 Dordrecht, the Netherlands: Martinus Nijhoff Publishers.

    Google Scholar 

  3. Awonaike KO, Danso SKA and Zapata F (1993) The use of a double isotope (15N and 34S) labelling technique to assess the suitability of various reference crops for estimating nitrogen fixation in Gliricidia sepium and Leucaena leucocephala. Plant and Sou 155/156: 325–328

    Article  Google Scholar 

  4. Ayanaba A and Lawson TL (1977) Diurnal changes in acetylene reduction in field-grown cowpeas and soybeans. Soil Biol Biochem 9: 125–129

    Article  CAS  Google Scholar 

  5. Boddey RM, Urquiaga S and Neves MR (1990) Quantification of the contribution of N2 fixation to field grown grain legumes — a strategy for the practical application of the 15N isotope dilution technique. Soil Biol Biochem 22: 649–655

    Article  CAS  Google Scholar 

  6. Chaiwanakupt P, Siripaibool C and Snitwongse P (1991) Evaluation of the appropriate non-N2-fixing crops to quantify nitrogen fixation by soybean using the 15N isotope dilution method. In: Proceedings Symposium on Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies, pp 89–99. STI/PUB/845, IAEA, Vienna

    Google Scholar 

  7. Chalk PM (1985) Estimation of N2 fixation by isotope dilution: An appraisal of techniques involving 15N enrichment and their application. Soil Biol Biochem 17: 389–410

    Article  CAS  Google Scholar 

  8. Danso SKA (1985) Methods of estimating biological nitrogen fixation. In: Ssali H and Keya SO (eds) Biological Nitrogen Fixation in Africa, pp 213–244. MIRCEN, Nairobi

    Google Scholar 

  9. Danso SKA (1988) The use of 15N enriched fertilizers for estimating nitrogen fixation in grain and pasture legumes. In: Beck DP and Materon LA (eds) Nitrogen Fixation by Legumes in Mediterranean Agriculture, pp 345–357 Dordrecht, the Netherlands: Martinus Nijhoff Publishers

    Google Scholar 

  10. Danso SKA (1991) Natural and artificial methods of 15N labelling of soil to estimate biological nitrogen fixation: Review of symposium papers. In: Proceedings Symposium on Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies pp 147–152. STI/PUB/845, IAEA, Vienna

    Google Scholar 

  11. Danso SKA, Bowen GD and Sanginga N (1992) Biological nitrogen fixation in trees in agro-ecosystem. Plant and Soil 141: 177–196

    Article  CAS  Google Scholar 

  12. Danso SKA, Hardarson G and Zapata F (1986) Assessment of dinitrogen fixation potentials of forage legumes with 15N technique. In: Haque I, Jutzi S, and Neate PJH (eds) Proceedings of a Workshop on Potentials of Forage Legumes in Farming Systems of Sub-Saharan Africa, pp 26–57. ILCA, Addis Ababa, Ethiopia

    Google Scholar 

  13. Danso SKA, Hardarson G and Zapata F (1988) Dinitrogen fixation estimates in alfalfa-ryegrass swards using different nitrogen-15 labelling methods. Crop Sci 28: 106–110

    Article  Google Scholar 

  14. Danso SKA, Hardarson G and Zapata F (1993) Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2fixation. Plant and Soil 152: 25–52

    Article  Google Scholar 

  15. Dommergues YR (1963) Evaluation du taux de fixation de l’azote dans un sol dunaire reboise filae (Casuarina equisetifolia). Agrochimica 105: 179–187

    CAS  Google Scholar 

  16. Denison RF, Weisz PR and Sinclair TR (1983) Analysis of acetylene reduction rates of soybean nodules at low acetylene concentrations. Plant Physiol 73: 648–651

    Article  CAS  Google Scholar 

  17. Dilworth M (1966) Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochem Bio-phys Acta 127: 285–294

    CAS  Google Scholar 

  18. Fried M and Broeshart H (1975) An independent measurement of the amount of nitrogen fixed by a legume crop. Plant and Soil 43: 707–711

    Article  Google Scholar 

  19. Fried M, Danso SKA and Zapata F (1983) The methodology of measurement of N2 fixation by non-legumes as inferred from field experiments with legumes. Can J Microbiol 29: 1053 –1062

    Article  Google Scholar 

  20. Fried M and Middelboe V (1977) Measurement of amount of nitrogen fixed by legume crop. Plant and Soil 43: 713–715

    Article  Google Scholar 

  21. Gauthier D, Diem HG, Dommergues YR and Ganry F (1985) Assessment of N2 fixation by Casuarina equisetifolia inoculated with Frankia ORSO21001 using 15N methods. Soil Biol Biochem 17: 375–380

    Article  CAS  Google Scholar 

  22. Ham GE (1978) Use of 15N in evaluating N2 fixation of field-grown soybeans. In Isotopes in Biological Dinitrogen Fixation, Proceedings Advisory Group Meeting, pp 151–161. STI/PUB/478, IAEA, Vienna

    Google Scholar 

  23. Hardarson G, Zapata F and Danso SKA (1988) Dinitrogen fixation measurements in alfalfa ryegrass swards using nitrogen-15 and influence of the reference crop. Crop Sci 28: 101–105

    Article  Google Scholar 

  24. Hardy RWF, Burns RC and Holsten RD (1973) Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol Biochem 5: 47–48

    Article  CAS  Google Scholar 

  25. Hardy RWF and Havelka UD (1975) Nitrogen fixation research — A key to world food? Science 188: 633–643

    Article  CAS  Google Scholar 

  26. Hardy RWF, Holsten RD, Jackson EK and Burns RC (1968) The acetylene-ethylene assay for N2 fixation: Laboratory and field evaluation. Plant Physiol 43: 1185–1207

    Article  CAS  Google Scholar 

  27. Herridge DF (1982a) Relative abundance of ureides and nitrate in plant tissues of soybean as a quantitative assay of nitrogen fixation. Plant Physiol 70: 1–6

    Article  CAS  Google Scholar 

  28. Herridge DF (1982b) Use of the ureide technique to describe the nitrogen economy of field-grown soybeans. Plant Physiol 70:7–11

    Article  CAS  Google Scholar 

  29. Herridge DF (1984) Effects of nitrate and plant development on the abundance of nitrogenous solutes in root-bleeding and vacuum-extracted exudates of soybean. Crop Sci 25: 173–179

    Article  Google Scholar 

  30. Kessel C van, Nakao P, Roskoski JP and Kevin K (1988) Ureide production by N2-fixing leguminous trees. Soil Biol Biochem 20: 891–897

    Article  Google Scholar 

  31. Knowles R (1981) The measurement of nitrogen fixation. In: Gibson AH and Newton WE (eds) Current Perspectives in Nitrogen Fixation, pp 26–57. Amsterdam: Elsevier Press

    Google Scholar 

  32. Kumarasinghe KS, Danso SKA and Zapata F (1992) Field evaluation of N2 fixation and partitioning in climbing bean (Phaseolus vulgaris L.) using 15N. Biol Fertil Soils 13: 142–146

    CAS  Google Scholar 

  33. McAuliffe C, Chamblee DS, Uribe-Arango H and Woodhouse Jr WW (1958) Influence of inorganic nitrogen on nitrogen fixation by legumes as revealed by 15N. Agron J 50: 334–337

    Article  CAS  Google Scholar 

  34. McClure PR and Israel DW (1979) Transport of nitrogen in the xylem of soybean plants. Plant Physiol 64: 411–416

    Article  CAS  Google Scholar 

  35. McClure PR, Israel DW and Volk RJ (1980) Evaluation of the relative ureide content of xylem sap as an indicator of N2 fixation in soybeans. Plant Physiol 66: 720–725

    Article  CAS  Google Scholar 

  36. Minchin FR, Sheehy JE and Witty JF (1986) Further errors in the acetylene reduction assay: effects of plant disturbance. J Exp Bot 37: 1581–1591

    Article  CAS  Google Scholar 

  37. Minchin FR, Witty JF and Mytton LR (1994) Reply to “Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay” by J.K. Vessey. Plant and Soil 158: 163–167

    Article  CAS  Google Scholar 

  38. Minchin FR, Sheehy JE and Muller M (1983) A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. J Exp Bot 34: 641–649

    Article  CAS  Google Scholar 

  39. Pate JS and Atkins CA (1983) Nitrogen uptake, transport and utilization. In: WJ Broughton (ed) Nitrogen Fixation Vol 3. Oxford: Clarendon Press

    Google Scholar 

  40. Patterson TG and LaRue TA (1983) Nitrogen fixation by soybeans: Seasonal and cultivar effects and comparison of estimates. Crop Sci 23: 488–492

    Article  Google Scholar 

  41. Peoples MB, Bergersen FJ, Turner GL, Sampet C, Rerkasen B, Bhromsiri A, Nurhayati DP, Faizah AW, Sudin MN, Norhayati M and Herridge DF (1991) Use of natural enrichment of 15N in plant available soil N for the measurement of symbiotic N2 fixation. In: Proceedings Symposium on Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies, pp 117–129 STI/PUB/845, IAEA, Vienna

    Google Scholar 

  42. Peoples MB, Faizah AW, Rerkasem B and Herridge DF (1989) Methods for Evaluating Nitrogen Fixation by Nodulated Legumes in the Field, Monograph No. 11. ACIAR, Canberra

    Google Scholar 

  43. Phillips DA, Jones MB, Center DM and Vaughn CE (1983) Estimating symbiotic nitrogen fixation by Trifolium subierraneum (cultivar Woogenellup) during regrowth. Agron J 75: 736–741

    Article  CAS  Google Scholar 

  44. Rainbird RM, Atkins CA and Pate JS (1983) Effect of temperature on nitrogenase functioning in cowpea nodules. Plant Physiol 73: 392–394

    Article  CAS  Google Scholar 

  45. Rennie RJ (1984) Comparison of N balance and 15N isotope dilution to quantify N2 fixation in field-grown legumes. Agron 176:785–790

    Google Scholar 

  46. Rennie RJ and Rennie DA (1983) Techniques for quantifying N2 fixation in association with non-legumes under field and greenhouse conditions. Can J Microbiol 29: 1022–1035

    Article  Google Scholar 

  47. Rennie RJ, Rennie DA and Fried M (1978) Concepts of 15N usage in dinitrogen fixation studies. In: Isotopes in Biological Dinitrogen Fixation, pp 107–130. IAEA, Vienna

    Google Scholar 

  48. Sanginga N, Danso SKA, Zapata F and Bowen GD (1990) Influence of reference trees on N2-fixation estimates in Leucaena leucocephala and Acacia albida using 15N labelling techniques. Biol Fertil Soils 9: 37–42

    Article  Google Scholar 

  49. Sanginga N, Mulongoy K and Ayanaba A (1988) Nodulation and growth of Leucaena leucocephala (Lam.) de Wit as affected by inoculation and N fertilizer. Plant and Soil 112: 129–135

    Article  CAS  Google Scholar 

  50. Shearer G and Kohl H (1986) N2-fixation in field settings: Estimations based on natural 15N abundance. Aust J Plant Physiol 13: 699–757

    CAS  Google Scholar 

  51. Stevenson FJ (1986) Nitrogen in Agricultural Soils. ASA Monograph No 22, 940 pp Madison, Wisconsin: American Society Agronomy

    Google Scholar 

  52. Streeter JG (1979) Allantoin and allantoic acid in tissues and stem exudate from field-grown soybean plant. Plant Physiol 63: 478–480

    Article  CAS  Google Scholar 

  53. Vessey JK (1994) Measurement of nitrogenase activity in legume root nodules. In: Defense of the Acetylene Reduction Assay. Plant and Soil 158: 151–162

    Google Scholar 

  54. Weaver RW (1986) Measurement of biological dinitrogen fixation in the field. In: Hauck RD and Weaver RW (eds) Field Measurement of Dinitrogen Fixation and Denitrification, pp 1–10. SSSA Special Publication No 18

    Google Scholar 

  55. Westermann DT, Kleinkopf GE, Porter LK and Leggett GE (1981) Nitrogen sources for bean production. Agron J 73: 660–664

    Article  CAS  Google Scholar 

  56. Witty JF (1983) Estimation of N2-fixation in the field using 15N-labelled fertilizer: Some problems and solutions. Soil Biol Biochem 15: 631–640

    Article  Google Scholar 

  57. Witty JF and Day JM (1978) Use of 15N2 in evaluation asymbiotic N2 fixation. In: Isotopes in Biological Dinitrogen Fixation. Proceedings Advisory Group Meeting, pp 135–150. STI/PUB/478, IAEA, Vienna

    Google Scholar 

  58. Witty JF and Minchin FR (1988) Measurement of nitrogen fixation by the acetylene reduction assay: myths and mysteries. In: Beck DP and Materon LA (eds) Nitrogen Fixation by Legumes in Mediterranean Agriculture, pp 331–344. Dordrecht: Martinus Nijhoff Publishers

    Google Scholar 

  59. Witty JF, Minchin FR, Sheehy JS and Minquez MI (1984) Acetylene-induced changes in the oxygen diffusion resistance and nitrogenase activity of legume root nodules. Ann Bot 53: 13–20

    CAS  Google Scholar 

  60. Witty JF and Ritz K (1984) Slow-release nitrogen-15 fertilizer formulations to measure nitrogen fixation by isotope dilution. Soil Biol Biochem 16: 657–661

    Article  CAS  Google Scholar 

  61. Zapata F, Danso SKA, Hardarson G and Fried M (1987a) Nitrogen fixation and translocation in field-grown fababean. Agron J 79: 505–509

    Article  Google Scholar 

  62. Zapata F, Danso SKA, Hardarson G and Fried M (1987b) Time course of nitrogen fixation in field-grown soybean using nitrogen-15 methodology. Agron J 79: 172–176

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. FAO/IAEA Division, Wagramerstrasse 5, P.O. Box 100, A-1400, Vienna, Austria

    S. K. A. Danso

Authors
  1. S. K. A. Danso
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. The University of the West Indies, St. Augustine, Trinidad W.I.

    N. Ahmad (Chairman, Organizing Committee) (Chairman, Organizing Committee)

Rights and permissions

Reprints and permissions

Copyright information

© 1995 Kluwer Academic Publishers

About this chapter

Cite this chapter

Danso, S.K.A. (1995). Assessment of biological nitrogen fixation. In: Ahmad, N. (eds) Nitrogen Economy in Tropical Soils. Developments in Plant and Soil Sciences, vol 69. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-1706-4_4

Download citation

  • DOI: https://doi.org/10.1007/978-94-009-1706-4_4

  • Publisher Name: Springer, Dordrecht

  • Print ISBN: 978-94-010-7264-9

  • Online ISBN: 978-94-009-1706-4

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation