, Volume 117, Issue 2–3, pp 359–373 | Cite as

Direct flux and 15N tracer methods for measuring denitrification in forest soils

  • Madhura V. Kulkarni
  • Amy J. Burgin
  • Peter M. GroffmanEmail author
  • Joseph B. Yavitt


Estimates of denitrification are one of the key uncertainties in the terrestrial nitrogen (N) cycle, primarily because reliable measurements of this highly variable process—especially the production of its terminal product (N2)—are difficult to obtain. We evaluated the ability of gas-flow soil core and 15N tracer methods to provide reliable estimates of denitrification in forest soils. Our objectives were to: (1) describe and present typical results from new gas-flow soil core and in situ 15N tracer methods for measuring denitrification, (2) discuss factors that affect the relevance of these methods to actual in situ denitrification, and (3) compare denitrification estimates produced by the two methods for a series of sites in a northern hardwood forest ecosystem. Both methods were able to measure accumulations of N2 over relatively short (2–5 h) incubations of either unamended or tracer-amended intact soils. Denitrification rates measured by the direct flux soil core method were very sensitive to incubation oxygen (O2) concentration and decreased with increased O2 levels. Denitrification rates measured by the in situ 15N tracer method were very sensitive to the 15N content of the nitrate (NO3 ) pool undergoing denitrification, which limits the applicability of this method for quantifying denitrification in N-poor ecosystems. While its ability to provide accurate estimates of denitrification was limited, the 15N tracer method provided estimates of the short-term abiotic and biotic transformations of atmospheric N deposition to gas. Furthermore, results suggest that denitrification is higher and that N2O:N2 ratios are lower (<0.02) than previously thought in the northern hardwood forest and that short-term abiotic and biotic transformations of atmospheric N deposition to gas are significant in this ecosystem.


Denitrification Forests Nitrogen 15Nitrate 



This research was supported by National Science Foundation grants DEB 0614158 and 0919047 (Ecosystem Studies) and DEB 0423259 (Hubbard Brook Long Term Ecological Research). The authors thank Lisa Martel, Dave Lewis and Stephanie Juice for help with field and laboratory analyses and two anonymous reviewers for helpful suggestions for revision.


  1. Bernal S, Hedin LO, Likens GE, Gerber S, Buso DC (2012) Complex response of the forest nitrogen cycle to climate change. Proc Natl Acad Sci USA 109(9):3406–3411CrossRefGoogle Scholar
  2. Bohme F, Russow R, Neue HU (2002) Airborne nitrogen input at four locations in the German state of Saxony-Anhalt—measurements using the 15N-based ITNI-system. Isot Environ Health Stud 38(2):95–102CrossRefGoogle Scholar
  3. Bormann BT, Bormann FH, Bowden WB, Pierce RS, Hamburg SP, Wang D, Snyder MC, Li CY, Ingersoll RC (1993) Rapid N2 fixation in pines, alder, and locust: evidence from the sandbox ecosystem study. Ecology 74(2):583–598CrossRefGoogle Scholar
  4. Bormann BT, Keller CK, Wang D, Bormann FH (2002) Lessons from the sandbox: is unexplained nitrogen real? Ecosystems 5(8):727–733CrossRefGoogle Scholar
  5. Bowden WB (1986) Gaseous nitrogen emissions from undisturbed terrestrial ecosystems—an assessment of their impacts on local and global nitrogen budgets. Biogeochemistry 2(3):249–279CrossRefGoogle Scholar
  6. Burgin AJ, Groffman PM (2012) Soil O2 controls denitrification rates and N2O yield in a riparian wetland. J Geophys Res Biogeosci 117(G1):G01010CrossRefGoogle Scholar
  7. Burgin AJ, Groffman PM, Lewis DN (2010) Factors regulating denitrification in a riparian wetland. Soil Sci Soc Am J 74(5):1826–1833CrossRefGoogle Scholar
  8. Butterbach-Bahl K, Willibald G, Papen H (2002) Soil core method for direct simultaneous determination of N2 and N2O emissions from forest soils. Plant Soil 240(1):105–116CrossRefGoogle Scholar
  9. Clough TJ, Stevens RJ, Laughlin RJ, Sherlock RR, Cameron KC (2001) Transformations of inorganic N in soil leachate under differing storage conditions. Soil Biol Biochem 33(11):1473–1480CrossRefGoogle Scholar
  10. Conley DJ, Paerl HW, Howarth RW, Boesch DF, Seitzinger SP, Havens KE, Lancelot C, Likens GE (2009) Controlling eutrophication: nitrogen and phosphorus. Science 323(5917):1014–1015CrossRefGoogle Scholar
  11. Crutzen PJ (1979) Role of NO and NO2 in the chemistry of the troposphere and stratosphere. Annu Rev Earth Planet Sci 7:443–472CrossRefGoogle Scholar
  12. Dannenmann M, Butterbach-Bahl K, Gasche R, Willibald G, Papen H (2008) Dinitrogen emissions and the N2:N2O emission ratio of a Rendzic Leptosol as influenced by pH and forest thinning. Soil Biol Biochem 40(9):2317–2323CrossRefGoogle Scholar
  13. Davidson EA (1992) Sources of nitric oxide and nitrous oxide following wetting of dry soil. Soil Sci Soc Am J 56(1):95–102CrossRefGoogle Scholar
  14. Davidson EA, Seitzinger S (2006) The enigma of progress in denitrification research. Ecol Appl 16(6):2057–2063CrossRefGoogle Scholar
  15. Davidson EA, David MB, Galloway JN, Goodale CL, Haeuber R, Harrison JA, Howarth RW, Jaynes DB, Lowrance RR, Nolan BT, Peel JL, Pinder RW, Porter E, Snyder CS, Townsend AR, Ward MH (2012) Excess nitrogen in the U.S. environment: trends, risks, and solutions. Issues Ecol 15:1–16Google Scholar
  16. Elliott EM, Kendall C, Wankel SD, Burns DA, Boyer EW, Harlin K, Bain DJ, Butler TJ (2007) Nitrogen isotopes as indicators of NOx source contributions to atmospheric nitrate deposition across the Midwestern and northeastern United States. Environ Sci Technol 41(22):7661–7667CrossRefGoogle Scholar
  17. Fuss C, Driscoll C, Johnson C, Petras R, Fahey T (2011) Dynamics of oxidized and reduced iron in a northern hardwood forest. Biogeochemistry 104(1):103–119CrossRefGoogle Scholar
  18. Galloway JN, Aber JD, Erisman JW, Seitzinger SP, Howarth RW, Cowling EB, Cosby BJ (2003) The nitrogen cascade. Bioscience 53(4):341–356CrossRefGoogle Scholar
  19. Galloway JN, Townsend AR, Erisman JW, Bekunda M, Cai ZC, Freney JR, Martinelli LA, Seitzinger SP, Sutton MA (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320(5878):889–892CrossRefGoogle Scholar
  20. Goodale CL, Aber JD, Vitousek PM (2003) An unexpected nitrate decline in New Hampshire streams. Ecosystems 6(1):75–86CrossRefGoogle Scholar
  21. Groffman P (2012) Terrestrial denitrification: challenges and opportunities. Ecol Process 1(1):11CrossRefGoogle Scholar
  22. Groffman PM, Altabet MA, Bohlke JK, Butterbach-Bahl K, David MB, Firestone MK, Giblin AE, Kana TM, Nielsen LP, Voytek MA (2006a) Methods for measuring denitrification: diverse approaches to a difficult problem. Ecol Appl 16(6):2091–2122CrossRefGoogle Scholar
  23. Groffman PM, Hardy JP, Driscoll CT, Fahey TJ (2006b) Snow depth, soil freezing, and fluxes of carbon dioxide, nitrous oxide and methane in a northern hardwood forest. Glob Change Biol 12(9):1748–1760CrossRefGoogle Scholar
  24. Hales HC, Ross DS (2008) Drastic short-term changes in the isotopic composition of soil nitrate in forest soil samples. Soil Sci Soc Am J 72(6):1645–1652CrossRefGoogle Scholar
  25. Hauck RD, Bouldin DR (1961) Distribution of isotopic nitrogen in nitrogen gas during denitrification. Nature 191:871–872CrossRefGoogle Scholar
  26. Hauck RD, Melsted SW, Yankwich PE (1958) Use of N-isotope distribution in nitrogen gas in the study of denitrification. Soil Sci 86:287–291CrossRefGoogle Scholar
  27. He CE, Liu XJ, Fangmeier A, Zhang FS (2007) Quantifying the total airborne nitrogen input into agroecosystems in the North China Plain. Agric Ecosyst Environ 121(4):395–400CrossRefGoogle Scholar
  28. Hofstra N, Bouwman A (2005) Denitrification in agricultural soils: summarizing published data and estimating global annual rates. Nutr Cycl Agroecosyst 72(3):267–278CrossRefGoogle Scholar
  29. Hogberg P (1997) 15N natural abundance in soil-plant systems. New Phytol 137(2):179–203CrossRefGoogle Scholar
  30. Howarth RW, Boyer EW, Pabich WJ, Galloway JN (2002) Nitrogen use in the United States from 1961–2000 and potential future trends. Ambio 31(2):88–96Google Scholar
  31. Judd KE, Likens GE, Groffman PM (2007) High nitrate retention during winter in soils of the Hubbard Brook Experimental Forest. Ecosystems 10(2):217–225CrossRefGoogle Scholar
  32. Kulkarni MV, Groffman PM, Yavitt JB (2008) Solving the global nitrogen problem: it’s a gas! Front Ecol Environ 6(4):199–206CrossRefGoogle Scholar
  33. Liptzin D, Silver W, Detto M (2011) Temporal dynamics in soil oxygen and greenhouse gases in two humid tropical forests. Ecosystems 14(2):171–182CrossRefGoogle Scholar
  34. Markfoged R, Nielsen LP, Nyord T, Ottosen LDM, Revsbech NP (2011) Transient N2O accumulation and emission caused by O2 depletion in soil after liquid manure injection. Eur J Soil Sci 62(4):541–550CrossRefGoogle Scholar
  35. Morse JL, Durán J, Groffman PM (2012) Seasonal patterns of denitrification and trace gas emissions in a northern hardwood forest. Abstract B53C-0682 presented at 2012 Fall Meeting, AGU, San Francisco, CA, 3–7 Dec 2012Google Scholar
  36. Mulvaney RL (1988) Evaluation of 15N tracer techniques for direct measurement of denitrification in soil III: laboratory studies. Soil Sci Soc Am J 52(5):1327–1332CrossRefGoogle Scholar
  37. Mulvaney RL, Vandenheuvel RM (1988) Evaluation of 15N tracer techniques for direct measurement of denitrification in soil IV: field studies. Soil Sci Soc Am J 52(5):1332–1337CrossRefGoogle Scholar
  38. Myrold DD (1990) Measuring denitrification in soils using 15N techniques. In: Revsbech NP, Sorenson J (eds) Denitrification in Soil and Sediment. Plenum Press, New York, pp 181–198CrossRefGoogle Scholar
  39. Parkin TB, Tiedje JM (1984) Application of a soil core method to investigate the effect of oxygen concentration on denitrification. Soil Biol Biochem 16(4):331–334CrossRefGoogle Scholar
  40. Parkin TB, Kaspar HF, Sexstone AJ, Tiedje JM (1984) A gas flow soil core method to measure field denitrification rates. Soil Biol Biochem 16(4):323–330CrossRefGoogle Scholar
  41. Parkin TB, Sexstone AJ, Tiedje JM (1985) Comparison of field denitrification rates determined by acetylene-based soil core and 15N methods. Soil Sci Soc Am J 49(1):94–99CrossRefGoogle Scholar
  42. Payne WJ (1981) Denitrification. Wiley, New YrokGoogle Scholar
  43. Payne WJ (1991) A review of methods for field measurements of denitrification. For Ecol Manag 44(1):5–14CrossRefGoogle Scholar
  44. Prather M, Derwent R, Ehhalt D, Fraser PJ, Sanhueza E, Zhou X (1995) Other trace gases and atmospheric chemistry. In: Houghton JT, Meiro Filho LG, Callander BA, Harris N, Kattenburg A and Maskell K (eds) Climate change 1994: radiative forcing of climate change and an evaluation of the IPCC IS92 emission scenarios. Cambridge University Press, New York. pp 73–126Google Scholar
  45. Raciti SR, Burgin AJ, Groffman PM, Lewis DN, Fahey TJ (2011) Denitrification in suburban lawn soils. J Environ Qual 40:1392–1940CrossRefGoogle Scholar
  46. Ravishankara AR, Daniel JS, Portmann RW (2009) Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326(5949):123–125CrossRefGoogle Scholar
  47. Robertson GP, Vitousek PM (2009) Nitrogen in agriculture: balancing the cost of an essential resource. Annu Rev Environ Res 34:97–125Google Scholar
  48. Roskoski JP (1980) Nitrogen fixation in hardwood forests of the northeastern United States. Plant Soil 54(1):33–44CrossRefGoogle Scholar
  49. Ryden JC, Skinner JH, Nixon DJ (1987) Soil core incubation system for the field measurement of denitrification using acetylene inhibition. Soil Biol Biochem 19(6):753–757CrossRefGoogle Scholar
  50. Schimel DS, Paul EA, Melillo J (1993) Theory and application of tracers (isotopic techniques in plant, soil, and aquatic biology). Academic Press, New YorkGoogle Scholar
  51. Scholefield D, Hawkins JMB, Jackson SM (1997a) Development of a helium atmosphere soil incubation technique for direct measurement of nitrous oxide and dinitrogen fluxes during denitrification. Soil Biol Biochem 29(9–10):1345–1352CrossRefGoogle Scholar
  52. Scholefield D, Hawkins JMB, Jackson SM (1997b) Use of a flowing helium atmosphere incubation technique to measure the effects of denitrification controls applied to intact cores of a clay soil. Soil Biol Biochem 29(9–10):1337–1344CrossRefGoogle Scholar
  53. Seitzinger S, Harrison JA, Bohlke JK, Bouwman AF, Lowrance R, Peterson B, Tobias C, Van Drecht G (2006) Denitrification across landscapes and waterscapes: a synthesis. Ecol Appl 16(6):2064–2090CrossRefGoogle Scholar
  54. Siegel RS, Hauck RD, Kurtz LT (1982) Determination of 30N2 and application to measurement of N2 evolution during denitrification. Soil Sci Soc Am J 46(1):68–74CrossRefGoogle Scholar
  55. Spott O, Stange CF (2007) A new mathematical approach for calculating the contribution of anammox, denitrification and atmosphere to an N-2 mixture based on a N-15 tracer technique. Rapid Commun Mass Spectrom 21(14):2398–2406CrossRefGoogle Scholar
  56. Spott O, Russow R, Apelt B, Stange CF (2006) A 15N-aided artificial atmosphere gas flow technique for online determination of soil N2 release using the zeolite Köstrolith SX6®. Rapid Commun Mass Spectrom 20(22):3267–3274CrossRefGoogle Scholar
  57. Stark JM, Hart SC (1996) Diffusion technique for preparing salt solutions, Kjeldahl digests, and persulfate digests for nitrogen-15 analysis. Soil Sci Soc Am J 60(6):1846–1855CrossRefGoogle Scholar
  58. Stehfest E, Bouwman L (2006) N2O and NO emission from agricultural fields and soils under natural vegetation: summarizing available measurement data and modeling of global annual emissions. Nutr Cycl Agroecosyst 74(3):207–228CrossRefGoogle Scholar
  59. Swerts M, Uytterhoeven G, Merckx R, Vlassak K (1995) Semicontinuous measurement of soil atmosphere gases with gas-flow soil core method. Soil Sci Soc Am J 59(5):1336–1342CrossRefGoogle Scholar
  60. Van Breemen N, Boyer EW, Goodale CL, Jaworski NA, Paustian K, Seitzinger SP, Lajtha K, Mayer B, Van Dam D, Howarth RW, Nadelhoffer KJ, Eve M, Billen G (2002) Where did all the nitrogen go? Fate of nitrogen inputs to large watersheds in the northeastern USA. Biogeochemistry 57(1):267–293CrossRefGoogle Scholar
  61. van Egmond K, Bresser T, Bouwman L (2002) The European nitrogen case. Ambio 31(2):72–78Google Scholar
  62. Venterea RT, Lovett GM, Groffman PM, Schwarz PA (2003) Landscape patterns of net nitrification in a northern hardwood-conifer forest. Soil Sci Soc Am J 67(2):527–539CrossRefGoogle Scholar
  63. Wang R, Willibald G, Feng Q, Zheng X, Liao T, Brüggemann N, Butterbach-Bahl K (2011) Measurement of N2, N2O, NO, and CO2 emissions from soil with the gas-flow-soil-core technique. Environ Sci Technol 45(14):6066–6072CrossRefGoogle Scholar
  64. Yang WH, Silver WL (2012) Application of the N2/Ar technique to measuring soil–atmosphere N2 fluxes. Rapid Commun Mass Spectrom 26(4):449–459CrossRefGoogle Scholar
  65. Yavitt JB, Fahey TJ, Simmons JA (1995) Methane and carbon dioxide dynamics in a northern hardwood ecosystem. Soil Sci Soc Am J 59(3):796–804CrossRefGoogle Scholar
  66. Zheng X, Fu C, Xu X, Yan X, Huang Y, Han S, Hu F, Chen G (2002) The Asian nitrogen cycle case study. Ambio 31(2):79–87Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Madhura V. Kulkarni
    • 1
    • 2
  • Amy J. Burgin
    • 3
  • Peter M. Groffman
    • 4
    Email author
  • Joseph B. Yavitt
    • 1
    • 2
  1. 1.Department of Natural ResourcesCornell UniversityIthacaUSA
  2. 2.Center for Integrative Natural Science and MathematicsNorthern Kentucky UniversityHighland HeightsUSA
  3. 3.School of Natural ResourcesUniversity of Nebraska-LincolnLincolnUSA
  4. 4.Cary Institute of Ecosystem StudiesMillbrookUSA

Personalised recommendations