Environmental Science and Pollution Research

, Volume 23, Issue 8, pp 7899–7910 | Cite as

Carbon amendment and soil depth affect the distribution and abundance of denitrifiers in agricultural soils

  • M. Barrett
  • M. I. Khalil
  • M. M. R. Jahangir
  • C. Lee
  • L. M. Cardenas
  • G. Collins
  • K. G. Richards
  • V. O’Flaherty
Research Article


The nitrite reductase (nirS and nirK) and nitrous oxide reductase-encoding (nosZ) genes of denitrifying populations present in an agricultural grassland soil were quantified using real-time polymerase chain reaction (PCR) assays. Samples from three separate pedological depths at the chosen site were investigated: horizon A (0–10 cm), horizon B (45–55 cm), and horizon C (120–130 cm). The effect of carbon addition (treatment 1, control; treatment 2, glucose-C; treatment 3, dissolved organic carbon (DOC)) on denitrifier gene abundance and N2O and N2 fluxes was determined. In general, denitrifier abundance correlated well with flux measurements; nirS was positively correlated with N2O, and nosZ was positively correlated with N2 (P < 0.03). Denitrifier gene copy concentrations per gram of soil (GCC) varied in response to carbon type amendment (P < 0.01). Denitrifier GCCs were high (ca. 107) and the bac:nirK, bac:nirS, bac:nir T , and bac:nosZ ratios were low (ca. 10−1/10) in horizon A in all three respective treatments. Glucose-C amendment favored partial denitrification, resulting in higher nir abundance and higher N2O fluxes compared to the control. DOC amendment, by contrast, resulted in relatively higher nosZ abundance and N2 emissions, thus favoring complete denitrification. We also noted soil depth directly affected bacterial, archaeal, and denitrifier abundance, possibly due to changes in soil carbon availability with depth.


Denitrification nirK nirS nosZ Subsoil N2O/N2 emissions 



The authors gratefully acknowledge the financial support for this study provided through the Research Stimulus Fund Programme of the Irish Department of Agriculture, Fisheries and Food (Grant RSF 06 383) and Science foundation Ireland (SFI) (2004/PI/416). The Authors would like to acknowledge Dr. D. Hatch and M. Butler at Rothamsted Research North Wyke Research, Devon, for all their help and support; Rothamsted Research is sponsored by the BBSRC, UK. Sincere thanks also to Dr. Laurent Phillipot and Dr. David Bru, for the gift of the nosZ plasmid (INRA-Université de Bourgogne, Soil and Environmental Microbiology, UMR MSE 1229, MSE, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France).


  1. Aber JD, Nadelhoffer KJ, Steudler P, Melillo JM (1989) Nitrogen Saturation in Northern Forest Ecosystems. Bioscience 39(6):378–386CrossRefGoogle Scholar
  2. Aber JD, McDowell W, Nadelhoffer K, Magill A, Berntson G, Kamakea M, McNulty S, Currie W, Rustad L, Fernandez I (1998) Nitrogen saturation in temperate forest ecosystems. Bioscience 48(11):921–934CrossRefGoogle Scholar
  3. Ågren GI, Bosatta E (1988) Nitrogen saturation of terrestrial ecosystems. Environ Pollut 54(3–4):185–197CrossRefGoogle Scholar
  4. Bergsma TT, Robertson GP, Ostrom NE (2002) Influence of soil moisture and land use history on denitrification end-products. J Environ Qual 31:711–717CrossRefGoogle Scholar
  5. Bouwman AF (2004) Direct emission of nitrous oxide from agricultural soils. Nutr Cycl Agroecosys 46(1):53–70CrossRefGoogle Scholar
  6. Boyer EW, Alexander RB, Parton WJ, Li C, Butterbach-Bahl K, Donner SD, Skaggs RW, Del Grosso SJ (2006) Modeling denitrification in terrestrial and aquatic ecosystems at regional scales. Ecol Appl 16:2123–2142CrossRefGoogle Scholar
  7. Braker G, Fesefeldt A, Witzel KP (1998) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ Microbiol 64:3769–3775Google Scholar
  8. Braker G, Zhou J, Wu L, Devol AH, Tiedje JM (2000) Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities. Appl Environ Microbiol 66:2096–2104CrossRefGoogle Scholar
  9. Cardenas LM, Hawkins JMB, Chadwick D, Scholefield D (2003) Biogenic gas emissions from soils measured using a new automated laboratory incubation system. Soil Biol Biochem 35:867–870CrossRefGoogle Scholar
  10. Carrigg C, Rice O, Kavanagh S, Collins G, O’Flaherty V (2007) DNA extraction method affects microbial community profiles from soils and sediment. Appl Microbiol Biotechnol 77(4):955–964CrossRefGoogle Scholar
  11. Casey RE, Taylor MD, Klaine SJ (2001) Mechanisms of nutrient attenuation in a subsurface flow riparian wetland. J Environ Qual 30(5):1732–1737CrossRefGoogle Scholar
  12. Cavigelli MA, Robertson GP (2000) The functional significance of denitrifier composition in an terrestrial ecosystem. Ecology 81:1401–1414CrossRefGoogle Scholar
  13. Cavigelli MA, Robertson GP (2001) Role of denitrifier diversity in rates of nitrous oxide consumption in a terrestrial ecosystem. Soil Biol Biochem 33:297–310CrossRefGoogle Scholar
  14. Chèneby D, Hartmann A, Hénault C, Topp E, Germon JC (1998) Diversity of denitrifying microflora and ability to reduce N2O in two soils. Biol Fert Soils 28:19–26CrossRefGoogle Scholar
  15. Clement JC, Pinay G, Marmonier P (2002) Seasonal dynamics of denitrification along topohydrosequences in three different riparian wetlands. J Environ Qual 31:1025–1037CrossRefGoogle Scholar
  16. Cofman AI, Levine JS (1986) Relative rates of nitric oxide and nitrous oxide production by nitrifiers, denitrifiers, and nitrate respirers. Appl Environ Microbiol 51(5):938–94Google Scholar
  17. Crutzen PJ (1970) The influence of nitrogen oxide on the atmospheric ozone content. Q J Roy Meteor Soc 96:320–327CrossRefGoogle Scholar
  18. Dandie CE, Burton DL, Zebarth BJ, Henderson SL, Trevors JT, Goyer C (2008) Changes in bacterial denitrifier community abundance over time in an agricultural field and their relationship with denitrification activity. Appl Environ Microbiol 74(19):5997–6005CrossRefGoogle Scholar
  19. Davidson EA, Seitzinger S (2006) The enigma of progress in denitrification research. Ecol Appl 16:2057–2063CrossRefGoogle Scholar
  20. De Vos P (2006) The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8(11):2012–2021CrossRefGoogle Scholar
  21. Deslippe JR, Jamali H, Jha N, Saggar S (2014) Denitrifier community size, structure and activity along a gradient of pasture to riparian soils. Soil Biol Biochem 71:48–60CrossRefGoogle Scholar
  22. Devaney D, Godley AR, Hodson ME, Purdy K, Yamulki S (2008) Impact of sewage sludge applications on the biogeochemistry of soils. Water Sci Technol 57(4):513–518CrossRefGoogle Scholar
  23. Dhondt K, Boeckx P, Hofman G, Van Cleemput O (2004) Temporal and spatial patterns of denitrification enzyme activity and nitrous oxide fluxes in three adjacent vegetated riparian buffer zones. Biol Fert Soils 40:243–251CrossRefGoogle Scholar
  24. Di HJ, Cameron KC, Sherlock RR, Shen J-P, He J-Z, Winefield CS (2010) Nitrous oxide emissions from grazed grassland as affected by a nitrification inhibitor, dicyandiamide, and relationships with ammonia-oxidizing bacteria and archaea. J Soils & Sediments 10(5):943–954CrossRefGoogle Scholar
  25. Dong LF, Smith CJ, Papaspyrou S, Stott A, Osborn M, Nedwell DB (2009) Changes in benthic denitrification, nitrate ammonification and anammox process rates and nitrate and nitrite reductase gene abundances along an estuarine nutrient gradient (the Colne Estuary, United Kingdom). Appl Environ Microbiol 75(10):3171–3179CrossRefGoogle Scholar
  26. Falk MW, Songa K-G, Matiasekb MG, Wuertz S (2009) Microbial community dynamics in replicate membrane bioreactors—natural reproducible fluctuations. Water Res 43:842–852CrossRefGoogle Scholar
  27. Flechard CR, Neftel A, JocherM AC, Fuhrer J (2005) Bi-directionalsoil/atmosphere N2O exchange over two mown grassland systems with contrasting management practices. Global Change Biol 11:2114–2127CrossRefGoogle Scholar
  28. Groffman PM, Altabet MA, Böhlke JK, Butterbach-Bahl K, David MB, Firestone MK, Giblin AE, Kana TM, Nielsen LP, Voytek MA (2006) Methods for measuring denitrification: diverse approaches to a difficult problem. Ecol Appl 16:2091–2122CrossRefGoogle Scholar
  29. Groffman PM, Butterbach-Bahl K, Fulweiler RW, Gold AJ, Morse JL, Stander EK, Tague C, Tonitto C, Vidon P (2009) Challenges to incorporating spatially and temporally explicit phenomena (hotspots and hot moments) in denitrification models. Biogeochemistry 93(1–2):49–77CrossRefGoogle Scholar
  30. Gruntzig V, Nold SC, Zhou J, Tiedje JM (2001) Pseudomonas stutzeri nitrate reductase gene abundance in environmental samples measured by real-time PCR. Appl Environ Microbiol 67:760–768CrossRefGoogle Scholar
  31. Hashimoto T, Whang KS, Nagaok K (2006) A quantitative evaluation and phylogenetic characterization of oligotrophic denitrifying bacteria harbored in subsurface upland soil using improved culturability. Biol Fert Soils 42:179–185CrossRefGoogle Scholar
  32. Henry S, Baudoin E, Lopez-Gutierrez JC, Martin-Laurent F, Brauman A, Philippot L (2004) Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J Microbiol Methods 59:327–335CrossRefGoogle Scholar
  33. Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative detection of the nosZ gene, encoding nitrous oxiden reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils. Appl Environ Microbiol 72(8):5181–5189CrossRefGoogle Scholar
  34. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012–2021CrossRefGoogle Scholar
  35. Jahangir MMR, Khalil MI, Johnston PM, Cardenas LM, Butler M, Hatch D, Barrett M, O’Flaherty V, Richards KG (2012) Denitrification potential in subsoils: a mechanism to reduce nitrate leaching to groundwater. Agric Ecosysts Environ 147:13–23CrossRefGoogle Scholar
  36. Jarvis SC, Hatch DJ (1994) Potential for denitrification at depth below long-term grass swards. Soil Biol Biochem 26(12):1629–1636CrossRefGoogle Scholar
  37. Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L (2006) Abundance of narG, nirS, nirK, and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland. Appl Environ Microbiol 72(9):5957–5962CrossRefGoogle Scholar
  38. Khalil MI, Richards KG (2011) Denitrification enzyme activity and potential of subsoils under grazed grasslands assayed by membrane inlet mass spectrometer. Soil Biol Biochem 43(9):1787–1797CrossRefGoogle Scholar
  39. Lee C, Kima J, Hwang K, O’Flaherty V, Hwang S (2009) Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters. Water Res 43(1):157–165CrossRefGoogle Scholar
  40. Ligi T, Oopkaup K, Truu M, Preem J-K, Nolvak H, Mitsch WJ, Mander Ü, Truu J (2014) Characterization of bacterial communities in soil and sediment of a created riverine wetland complex using high-throughput 16 S rRNA amplicon sequencing. Ecol Eng 72:56–66CrossRefGoogle Scholar
  41. Lovely DR, Chapelle FH (1995) Deep subsurface microbial processes. Rev Geophys 33(3):365–381CrossRefGoogle Scholar
  42. Martin TL, Kaushik NK, Trevors JT, Whiteley JR (1999) Review: Denitrification in temperate climate riparian zones. Water Air Soil Pollut 111(1–4):171–186CrossRefGoogle Scholar
  43. McCarty GW, Bremner JM (1992) Availability of organic carbon for denitrification of nitrate in subsoils. Biol Fert Soils 14(3):219–222CrossRefGoogle Scholar
  44. McCarty GW, Bremner JM (1993) Factors affecting the availability of organic carbon for denitrification of nitrate in subsoils. Biol Fert Soils 15(2):132–136CrossRefGoogle Scholar
  45. McCune B, Grace JB (2002) Analysis of ecological communities, pg 1–218. Published by MjM Software Design in Gleneden Beach, ORGoogle Scholar
  46. Mei L, Yang L, Wang D, Yin B, Hu J, Yina S (2004) Nitrous oxide production and consumption in serially diluted soil suspensions as related to in situ N2O emission in submerged soils. Soil Biol Biochem 36:1057–1066CrossRefGoogle Scholar
  47. Melero S, Pérez-de-Mora A, Murillo J-M, Buegger F, Kleinedamb K, Kublik S, Vanderlinden K, Moreno F, Schloter M (2011) Denitrification in a vertisol under long-term tillage and no-tillage management in dryland agricultural systems: Key genes and potential rates. Appl Soil Ecol 47(3):221–225CrossRefGoogle Scholar
  48. Morley NJ, Richardson DJ, Baggs EM (2014) Substrate induced denitrification over or under estimates shifts in soil N2/N2O ratios. PLoS ONE 9(9)Google Scholar
  49. Oehler F, Bordenave P, Durand P (2007) Variations of denitrification in a farming catchment area. Agric Ecosyst Environ 120(2–4):313–324CrossRefGoogle Scholar
  50. Osaka T, Shirotani K, Yoshie S, Tsuneda S (2008) Effects of carbon source on denitrification efficiency and microbial community structure in a saline wastewater treatment process. Water Res 42:3709–3718CrossRefGoogle Scholar
  51. Peralta AL, Matthews JWD, Kent JW (2010) Microbial community structure and denitrification in a wetland mitigation bank. Appl Environ Microbiol 76:4207–4215CrossRefGoogle Scholar
  52. Priemé A, Braker G, Tiedje JM (2002) Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils. Appl Environ Microbiol 68:1893–1900CrossRefGoogle Scholar
  53. Rangeley A, Knowles R (1988) Nitrogen transformations in a Scottish peat soil under laboratory conditions. Soil Biol Biochem 20:385–391CrossRefGoogle Scholar
  54. Rasmussen R (2001) Quantification on the LightCycler. In: Meuer S, Wittwer C, Nakagawara K (eds) Rapid cycle real-time PCR: methods and applications. Springer, BerlinGoogle Scholar
  55. Richardson D, Felgate H, Watmough N, Thomson A, Baggs E (2009) Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle—could enzymic regulation hold the key? Trends Biotechnol 27(7):388–397CrossRefGoogle Scholar
  56. Robertson GP, Groffman PM (2007) Nitrogen transformation. In: Paul EA (ed) Soil Microbiology, Biochemistry, and Ecology. Springer, New York, pp 341–364Google Scholar
  57. Rypdal K, Winiwarter K (2001) Uncertainties in greenhouse gas inventories—evaluation, comparability and implications. Environ Sci Policy 4:107–116CrossRefGoogle Scholar
  58. Scala DJ, Kerkhof LJ (1998) Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments. FEMS Microbiol Lett 162:61–68CrossRefGoogle Scholar
  59. Scholefield D, Hawkins JMB, Jackson SM (1997a) A.). Development of a helium atmosphere soil incubation technique for direct measurement of nitrous oxide and dinitrogen fluxes during denitrification. Soil Biol Biochem 29:1345–1352CrossRefGoogle Scholar
  60. Scholefield D, Hawkins JMB, Jackson SM (1997b) B.). Use of a flowing helium atmosphere incubation technique to measure the effects of denitrification controls applied to intact soil cores of a clay soil. Soil Biol Biochem 29:1337–1344CrossRefGoogle Scholar
  61. Sotomayor D, Rice CW (1996) Denitrification in soil profiles beneath grassland and cultivated soils. Soil Sci Am J 60(6):1822–1828CrossRefGoogle Scholar
  62. Stark C, Richards K (2008) The continuing challenge of agricultural nitrogen loss to the environment in the context of global change and advancing research. Dyn Soil, Dyn Plant 2(1):1–12Google Scholar
  63. Tatti E, Goyer C, Burton DL, Wertz S, Zebarth BJ, Chantigny M, Filion M (2015) Tillage management and seasonal effects on denitrifier community abundance, gene expression and structure over winter. Microb Ecol 70(3):795–808CrossRefGoogle Scholar
  64. Throbäck IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:401–417CrossRefGoogle Scholar
  65. Tiedje JM (1988) Ecology of denitrification and dissimilatory nitrate reduction to ammonium. In: Zehnder A (ed) Biology of anaerobic microorganisms. Wiley, New York, N.Y., pp 179–244Google Scholar
  66. Vilain G, Garnier J, Decuq C, Lugnot M (2014) Nitrous oxide production from soil experiments: denitrification prevails over nitrification. Nutr Cycl Agroecosyst 98:169–186CrossRefGoogle Scholar
  67. Whelan JA, Russel NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real time PCR. J Immunol Methods 278:261–269CrossRefGoogle Scholar
  68. Wrage N, Velthof GL, van Beusichem ML, Oenema O (2001) Role of nitrifier denitrification in the production of nitrous oxide. Soil Biol Biochem 33(12–13):1723–1732CrossRefGoogle Scholar
  69. Yu Y, Lee C, Kim J, Hwang S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89(6):670–679CrossRefGoogle Scholar
  70. Zumft WG (1992) The denitrifying prokaryotes, In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH, (eds) The Prokaryotes, 2nd edn. Springer-Verlag, New York, pp 554–582Google Scholar
  71. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • M. Barrett
    • 1
  • M. I. Khalil
    • 2
  • M. M. R. Jahangir
    • 2
  • C. Lee
    • 4
  • L. M. Cardenas
    • 5
  • G. Collins
    • 3
    • 6
  • K. G. Richards
    • 2
  • V. O’Flaherty
    • 1
  1. 1.Microbial Ecology Laboratory, Microbiology, School of Natural Sciences & Ryan InstituteNational University of Ireland GalwayGalwayIreland
  2. 2.Crops, Environment and Land-Use Department, Teagasc, Johnstown Castle, Co.WexfordIreland
  3. 3.Microbial EcoEngineering Laboratory, Microbiology, School of Natural Sciences & Ryan InstituteNational University of Ireland GalwayGalwayIreland
  4. 4.School of Civil & Environmental Engineering Nanyang Technological UniversitySingaporeSingapore
  5. 5.Rothamsted Research, North WykeOkehamptonUK
  6. 6.Environmental Engineering Laboratory, School of EngineeringUniversity of GlasgowGlasgowUK

Personalised recommendations