Antonie van Leeuwenhoek

, Volume 110, Issue 11, pp 1453–1465 | Cite as

Denitrifying bacterial communities display different temporal fluctuation patterns across Dutch agricultural soils

  • Nguyen E. López-Lozano
  • Michele C. Pereira e SilvaEmail author
  • Franck Poly
  • Nadine Guillaumaud
  • Jan Dirk van Elsas
  • Joana Falcão Salles
Original Paper


Considering the great agronomic and environmental importance of denitrification, the aim of the present study was to study the temporal and spatial factors controlling the abundance and activity of denitrifying bacterial communities in a range of eight agricultural soils over 2 years. Abundance was quantified by qPCR of the nirS, nirK and nosZ genes, and the potential denitrification enzyme activity (DEA) was estimated. Our data showed a significant temporal variation considerably high for the abundance of nirK-harboring communities, followed by nosZ and nirS communities. Regarding soil parameters, the abundances of nosZ, nirS and nirK were mostly influenced by organic material, pH, and slightly by NO3 , respectively. Soil texture was the most important factor regulating DEA, which could not be explained by the abundance of denitrifiers. Analyses of general patterns across lands to understand the soil functioning is not an easy task because the multiple factors influencing processes such as denitrification can skew the data. Careful analysis of atypical sites are necessary to classify the soils according to trait similarity and in this way reach a better predictability of the denitrifiers dynamics.


Gene abundance nosnirnirPotential denitrification Soil texture 



This work was supported by the NWO-ERGO Program and was part of a collaborative project with Utrecht University, Utrecht, The Netherlands. We would like to thank our colleagues Alexander V Semenov and Jolanda Brons, for their help with sampling. NELL thanks to F.R. Beamonte-Barrientos for the advices in the statistical analysis.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10482_2017_898_MOESM1_ESM.docx (61 kb)
Supplementary material 1 (DOCX 60 kb)
10482_2017_898_MOESM2_ESM.docx (1.2 mb)
Supplementary material 2 (DOCX 1223 kb)


  1. Akiyama H, Yan X, Yagi K (2010) Evaluation of effectiveness of enhanced-efficiency fertilizers as mitigation options for N2O and NO emissions from agricultural soils: meta-analysis. Global Change Biol 16:1837–1846Google Scholar
  2. Attard E, Recous S, Chabbi A, De Berranger C, Guillaumaud N, Labreuche J, Philippot L, Schmid B, Le Roux X (2011) Soil environmental conditions rather than denitrifier abundance and diversity drive potential denitrification after changes in land uses. Global Change Biol 17:1975–1989CrossRefGoogle Scholar
  3. Baggs EM, Smales CL, Bateman EJ (2010) Changing pH shifts the microbial source as well as the magnitude of N2O emission from soil. Biol Fertil Soils 46:793–805CrossRefGoogle Scholar
  4. Barrett M, Khalil MI, Jahangir MMR, Lee C, Cardenas LM, Collins G, Richards KG, O’Flaherty V (2016) Carbon amendment and soil depth affect the distribution and abundance of denitrifiers in agricultural soils. Environ Sci Pollut Res 23(8):7899–7910. doi: 10.1007/s11356-015-6030-1
  5. Bateman EJ, Baggs EM (2005) Contributions of nitrification and denitrification to N2O emissions from soils at different water-filled pore space. Biol Fertil Soils 41:379–388CrossRefGoogle Scholar
  6. Baudoin E, Philippot L, Cheneby D, Chapuis-Lardy L, Fromin N, Bru D et al (2009) Direct seeding mulch-based cropping increases both the activity and the abundance of denitrifier communities in a tropical soil. Soil Biol Biochem 4:1703–1709Google Scholar
  7. Burton DL, Zebarth BJ, McLeod JA, Goyer C (2012) Nitrous oxide emissions from potato production and strategies to reduce them. Sustain Potato Prod 4:251–271Google Scholar
  8. Butler C, Richardson D (2005) The emerging molecular structure of the nitrogen cycle: an introduction to the proceedings of the 10th annual N-cycle meeting. Biochem Soc T 33:113–118CrossRefGoogle Scholar
  9. Butterbach-Bahl K, Baggs EM, Dannenmann M, Kiese R, Zechmeister-Boltenstern S (2013) Nitrous oxide emissions from soils: how well do we understand the processes and their controls? Phil Trans R Soc B 368:20130122CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chroňáková A, Radl V, Čuhel J, Šimek M, Elhottová D, Engel M, Schloter M (2009) Overwintering management on upland pasture causes shifts in an abundance of denitrifying microbial communities, their activity and N2O-reducing ability. Soil Biol Biochem 41:1132–1138CrossRefGoogle Scholar
  11. Clément JC, Pinay G, Marmonier P (2002) Seasonal dynamics of denitrification along topohydrosequences in three different riparian wetlands. J Environ Qual 31:1025–1037CrossRefPubMedGoogle Scholar
  12. Cuhel J, Simek M, Laughlin RJ, Bru D, Chèneby D, Watson CJ, Philippot L (2010) Insights into the effect of soil pH on N2O and N2 emissions and denitrifier community size and activity. Appl Environ Microb 76:1870–1878CrossRefGoogle Scholar
  13. D’Haene K, Moreels E, De Neve S, Chaves DB, Boeckx P, Hofman G, Van Cleemput O (2003) Soil properties influencing the denitrification potential of Flemish agricultural soils. Biol Fertil Soils 38:358–366CrossRefGoogle Scholar
  14. 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 Microb 74:5997–6005CrossRefGoogle Scholar
  15. Dandie CE, Wertz S, Leclair CL, Goyer C, Burton DL, Patten CL, Zebarth BJ, Trevors JT (2011) Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones. FEMS Microbiol Ecol 77:69–82CrossRefPubMedGoogle Scholar
  16. Djigal D, Baudoin E, Philippot L, Brauman A, Villenave C (2010) Shifts in size, genetic structure and activity of the soil denitrifier community by nematode grazing. Eur J Soil Biol 46:112–118CrossRefGoogle Scholar
  17. Dong LF, Smith CJ, Papaspyrou S, Stott A, Osborn AM, 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 75:3171–3179CrossRefPubMedPubMedCentralGoogle Scholar
  18. Dorland S, Beauchamp EG (1991) Denitrification and ammonification at low soil temperatures. Can J Soil Sci 71(3):293–303CrossRefGoogle Scholar
  19. Enwall K, Throbäck IN, Stenberg M, Söderström M, Hallin S (2010) Soil resources influence spatial patterns of denitrifying communities at scales compatible with land management. Appl Environ Microbiol 76:2243–2250CrossRefPubMedPubMedCentralGoogle Scholar
  20. Firestone MK, Davidson EA (1989) Microbiological basis of NO and N2O production and consumption in soil. In: Exchange of trace gases between terrestrial ecosystems and the atmosphere, vol 47, pp 7–21Google Scholar
  21. Glockner AB, Jüngst A, Zumft WG (1993) Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS-) of Pseudomonas stutzeri. Arch Microbiol 160:18–26Google Scholar
  22. Green CT, Böhlke JK, Bekins BA, Phillips SP (2010) Mixing effects on apparent reaction rates and isotope fractionation during denitrification in a heterogeneous aquifer. Water Resour Res 46(8). doi: 10.1029/2009WR008903
  23. Groffman P, Holland E, Myrold DD, Robertson GP, Zou X (1999) Denitrification. In: Robertson GP, Coleman DC, Bledoe CS, Sollins P (eds) Standard soil methods for long-term ecological research. Oxford University Press, Oxford, pp 272–288Google Scholar
  24. Gu C, Riley WJ (2010) Combined effects of short term rainfall patterns and soil texture on soil nitrogen cycling—a modeling analysis. J Contam Hydrol 112:141–154CrossRefPubMedGoogle Scholar
  25. Gu J, Nicoullaud B, Rochette P, Grossel A, Hénault C, Cellier P, Richard G (2013) A regional experiment suggests that soil texture is a major control of N2O emissions from tile-drained winter wheat fields during the fertilization period. Soil Biol Biochem 60:134–141CrossRefGoogle Scholar
  26. Hallin S, Jones CM, Schloter M, Philippot L (2009) Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment. ISME J 3:597–605CrossRefPubMedGoogle Scholar
  27. Henderson SL, Dandie CE, Patten CL, Zebarth BJ, Burton DL, Trevors JT, Goyer C (2010) Changes in denitrifier abundance, denitrification gene mRNA levels, nitrous oxide emissions, and denitrification in anoxic soil microcosms amended with glucose and plant residues. Appl Environ Microbiol 76:2155–2164CrossRefPubMedPubMedCentralGoogle Scholar
  28. Henry S, Baudoin E, López-Gutiérrez JC, Martin-Laurent F, Brauman A, Laurent Philippot (2004) Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR. J Microbiol Meth 59:327–335CrossRefGoogle Scholar
  29. Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils. Appl Environ Microb 72:5181–5189CrossRefGoogle Scholar
  30. Henry S, Texier S, Hallet S, Bru D, Dambreville C, Chèneby D, Bizouard F, Germon JC, Philippot L (2008) Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: insight into the role of root exudates. Environ Microbiol 10:2092–3082CrossRefGoogle Scholar
  31. Hofstra N, Bouwman AF (2005) Denitrification in agricultural soils: summarizing published data and estimating global annual rates. Nutr Cycl Agroecosyst 72:267–278CrossRefGoogle Scholar
  32. Jones C, Hallin S (2010) Ecological and evolutionary factors underlying global and local assembly of denitrifier communities. ISME J 4:633–641CrossRefPubMedGoogle Scholar
  33. Jones CM, Welsh A, Throbäck IN, Dörsch P, Bakken LR, Hallin S (2011) Phenotypic and genotypic heterogeneity among closely related soil-borne N2- and N2O-producing Bacillus isolates harboring the nosZ gene. FEMS Microbiol Ecol 76:541–552CrossRefPubMedGoogle Scholar
  34. 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 72:5957–5962CrossRefPubMedPubMedCentralGoogle Scholar
  35. Knowles R (1982) Denitrification. Microbiol Rev 46:43Google Scholar
  36. Ligi LT, Truu M, Truu J, Nõlvak H, Kaasik A, Mitsch WJ, Mander Ü (2014) Effects of soil chemical characteristics and water regime on denitrification genes (nirS, nirK, and nosZ) abundances in a created riverine wetland complex. Ecol Eng 72:47–55CrossRefGoogle Scholar
  37. Liu X, Tiquia SM, Holguin G, Wu L, Nold SC, Devol AH, Luo K, Palumbo AV, Tiedje JM, Zhou J (2003) Molecular diversity of denitrifying genes in continental margin sediments within the oxygen-deficient zone off the Pacific Coast of Mexico. Appl Environ Microbiol 69:3549–3560CrossRefPubMedPubMedCentralGoogle Scholar
  38. López-Lozano NE, Eguiarte LE, Bonilla-Rosso G, García-Oliva F, Martínez-Piedragil C, Rooks C, Souza V (2012) Bacterial communities and the nitrogen cycle in the gypsum soils of Cuatro Ciénegas Basin, Coahuila: a Mars analogue. Astrobiology 12:699–709CrossRefPubMedPubMedCentralGoogle Scholar
  39. Marton JM, Chowdhury RR, Craft CB (2015) A comparison of the spatial variability of denitrification and related soil properties in restored and natural depression wetlands in Indiana, USA. Int J Biodiv Sci Ecosyst Serv Manag 11:36–45CrossRefGoogle Scholar
  40. McGill BM, Sutton-Grier AE, Wright JP (2010) Plant trait diversity buffers variability in denitrification potential over changes in season and soil conditions. PLoS ONE 5:e11618CrossRefPubMedPubMedCentralGoogle Scholar
  41. Mergel A, Schmitz O, Mallmann T, Bothe H (2001) Relative abundance of denitrifying and dinitrogen-fixing bacteria in layers of a forest soil. FEMS Microbiol Ecol 36:33–42CrossRefPubMedGoogle Scholar
  42. Miller MN, Zebarth BJ, Dandie CE, Burton DL, Goyer C, Trevors JT (2008) Crop residue influence on denitrification, N2O emissions and denitrifier community abundance in soil. Soil Biol Biochem 40:2553–2562CrossRefGoogle Scholar
  43. Mørkved PT, Dörsch P, Bakken LR (2007) The N2O product ratio of nitrification and its dependence on long-term changes in soil pH. Soil Biol Biochem 39:2048–2057CrossRefGoogle Scholar
  44. Morse JL, Ardón M, Bernhardt ES (2012) Using environmental variables and soil processes to forecast denitrification potential and nitrous oxide fluxes in coastal plain wetlands across different land uses. J Geophys Res: Biogeosci 117(G2). doi: 10.1029/2011JG001923
  45. Mosier AR, Doran JW, Freney JR (2002) Managing soil denitrification. J Soil Water Conserv 57:505–512Google Scholar
  46. Niboyet A, Barthes L, Hungate BA, Le Roux X, Bloor JMG, Ambroise A, Fontaine S, Price PM, Leadley PW (2009) Responses of soil nitrogen cycling to the interactive effects of elevated CO2 and inorganic N supply. Plant Soil 327:35–47CrossRefGoogle Scholar
  47. Patra AK, Abbadie L, Clays-Josserand A, Degrangeet V, Grayston SJ, Loiseau P, Louault F, Mahmood S, Nazaret S, Philippot L, Poly F, Prosser JI, Richaume A, Le Roux X (2005) Effects of grazing on microbial functional groups involved in soil N dynamics. Ecol Monogr 75:65–80CrossRefGoogle Scholar
  48. Pereira e Silva MC, Dias ACF, van Elsas JD, Salles JF (2012) Spatial and temporal variation of archaeal, bacterial and fungal communities in agricultural soils. PLoS ONE 7:e51554CrossRefPubMedPubMedCentralGoogle Scholar
  49. Petersen DG, Blazewicz SJ, Firestone M, Herman DJ, Turetsky M, Waldrop M (2012) Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska. Environ Microbiol 14:993–1008CrossRefPubMedGoogle Scholar
  50. Philippot L, Cuhel J, Saby NP, Chèneby D, Chronáková A, Bru D, Arrouays D, Martin-Laurent F, Šimek M (2009) Mapping field-scale spatial patterns of size and activity of the denitrifier community. Environ Microbiol 11:1518–1526CrossRefPubMedGoogle Scholar
  51. R Development Core Team (2008) R: a language and environment for statistical computing. R Foundation for Statistical Computing ISBN 3–900051–07–0.
  52. Rasche F, Knapp D, Kaiser C, Koranda M, Kitzler B, Zechmeister-Boltenstern S, Richter A, Sessitsch A (2011) Seasonality and resource availability control bacterial and archaeal communities in soils of a temperate beech forest. ISME J 5:389–402CrossRefPubMedGoogle Scholar
  53. Rosa SM, Kraemer FB, Soria MA, Guerrero LD, Morrás HJM, Figuerola ELM, Erijman L (2014) The influence of soil properties on denitrifying bacterial communities and denitrification potential in no-till production farms under contrasting management in the Argentinean Pampas. Appl Soil Ecol 75:172–180CrossRefGoogle Scholar
  54. Šimek M, Cooper JE (2002) The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years. Eur J Soil Sci 53:345–354CrossRefGoogle Scholar
  55. Smith JM, Ogram A (2008) Genetic and functional variation in denitrifier populations along a short-term restoration chronosequence. Appl Environ Microbiol 74:5615–5620CrossRefPubMedPubMedCentralGoogle Scholar
  56. Smith MS, Tiedje JM (1979) Phases of denitrification following oxygen depletion in soil. Soil Biol Biochem 11:261–267CrossRefGoogle Scholar
  57. Smith KA, McTaggart IP, Dobbie KE, Conen F (1998) Emissions of N2O from Scottish soils, as a function of fertilizer N. Nutr Cycl Agroecosyst 52:123–130CrossRefGoogle Scholar
  58. Song K, Kang H, Zhang L, Mitsch WJ (2012) Seasonal and spatial variations of denitrification and denitrifying bacterial community structure in created riverine wetlands. Ecol Eng 38:130–134CrossRefGoogle Scholar
  59. Strauss EA, Richardson WB, Cavanaugh JC, Bartsch LA, Kreiling RM, Standorf AJ (2006) Variability and regulation of denitrification in an Upper Mississippi River backwater. J N Am Benthol Soc 25:596–606CrossRefGoogle Scholar
  60. Syakila A, Kroeze C (2011) The global nitrogen budget revisited. Greenhouse Gas Meas Manage 1:17–26. doi: 10.3763/ghgmm.2010.0007 CrossRefGoogle Scholar
  61. Throbäck I, fEnwall 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 49:401–417CrossRefPubMedGoogle Scholar
  62. Van den Heuvel RN, Bakker SE, Jetten MSM, Hefting MM (2011) Decreased N2O reduction by low soil pH causes high N2O emissions in a riparian ecosystem. Geobiol 9:294–300Google Scholar
  63. Wallenstein MD, Peterjohn WT, Schlesinger WH (2006) N fertilization effects on denitrification and n cycling in an aggrading forest. Ecol Appl 16:2168–2176CrossRefPubMedGoogle Scholar
  64. Yoshida M, Ishii S, Otsuka S, Senoo K (2010) nirK-harboring denitrifiers are more responsive to denitrification- inducing conditions in rice paddy soil than nirS-harboring bacteria. Microbes Environ 25:45–48CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Department of Microbial Ecology, Centre for Life SciencesUniversity of GroningenGroningenThe Netherlands
  2. 2.División de Ciencias AmbientalesInstituto Potosino de Investigación Científica y Tecnológica (IPICyT)San Luis PotosíMexico
  3. 3.Microbial Ecology Centre (UMR 5557 CNRS-Université Lyon 1; USC 1193 INRA)VilleurbanneFrance
  4. 4.Soil Microbiology Laboratory, Soil Science Department, College of Agriculture “Luiz de Queiroz”University of Sao PauloSão PauloBrazil

Personalised recommendations