Environmental Science and Pollution Research

, Volume 20, Issue 9, pp 6646–6657 | Cite as

Abundance of denitrification genes under different peizometer depths in four Irish agricultural groundwater sites

  • Maria Barrett
  • Mohammad M. R. Jahangir
  • Changsoo Lee
  • Cindy J. Smith
  • Niamh Bhreathnach
  • Gavin Collins
  • Karl G. Richards
  • Vincent O’Flaherty
Research Article


This study examined the relationship between the abundance of bacterial denitrifiers in groundwater at four sites, differing with respect to overlaying land management and peizometer depth. Groundwater was sourced from 36 multilevel piezometers, which were installed to target different groundwater zones: (1) subsoil, (2) subsoil to bedrock interface, and (3) bedrock. The gene copy concentrations (GCCs), as gene copies per liter, for bacterial 16S rRNA genes and the denitrifying functional genes, nirK, nirS, and nosZ, were determined using quantitative polymerase chain reaction assays. The results were related to gaseous nitrogen emissions and to the physicochemical properties of the four sites. Overall, nirK and nirS abundance appeared to show no significant correlation to N2O production (P = 0.9989; P = 0.3188); and no significant correlation was observed between nosZ and excess N2 concentrations (P = 0.0793). In the majority of piezometers investigated, the variation of nirK and nirS gene copy concentrations was considered significant (P < 0.0001). Dissolved organic carbon (DOC) decreased with aquifer depth and ranged from 1.0–4.0 mg l−1, 0.9–2.4 mg l−1, and 0.8–2.4 mg l−1 within piezometers located in the subsoil, subsoil/bedrock interface, and bedrock depths, respectively. The availability of increasing DOC and the depth of the water table were positively correlated with increasing nir and nosZ GCCs (P = 0.0012). A significant temporal correlation was noted between nirS and piezometer depth (P < 0.001). Interestingly, the nirK, nirS, and nosZ GCCs varied between piezometer depths within specific sites, while GCCs remained relatively constant from site to site, thus indicating no direct impact of agricultural land management strategies investigated on denitrifier abundance.


Denitrification Subsoil Piezometer Groundwater nirK nirS nosZ qPCR 


  1. Addy K, Gold AJ, Nowicki B, McKenna J, Stolt M, Groffman PM (2005) Denitrification capacity in a subterranean estuary below a Rhode Island fringing salt marsh. Estuaries 29:896–908CrossRefGoogle Scholar
  2. Aggelopoulos CA, Tsakiroglou CD (2009) A multi-flowpath model for the interpretation of immiscible displacement experiments in heterogeneous soil columns. J Contam Hydrol 105:146–160CrossRefGoogle Scholar
  3. Andraski TW, Bundy LG, Brye KR (2000) Crop management and corn nit rogen rate effects on nitrate leaching. J Environ Qual 29:1095–1103CrossRefGoogle Scholar
  4. Bilanovic D, Battistoni P, Cecchi F, Pavan P, Mataalvarez J (1999) Denitrification under high nitrate concentration and alternating anoxic conditions. Water Res 33(15):3311–3320CrossRefGoogle Scholar
  5. Bohlke KJ, Wanty R, Tuttle M, Delin G, Landon M (2002) Denitrification in the recharge and discharge area of a transient agricultural nitrate plume in a glacial outwash sand aquifer. Minnesota. Water Res Res 38:1–26CrossRefGoogle Scholar
  6. Bouwman AF, Van Drecht G, van der Hoek KW (2005) Surface N balances and reactive N loss to the environment from global intensive agricultural production systems for the period 1970–2030. Sci China Life Sci 48:113Google Scholar
  7. Braker G, Fesefeldt A, Witzel K-P (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. Butturini A, Bernal S, Nin E, Hellin C, Rivero L, Sabater S, Sabater F (2003) Influences of the stream groundwater hydrology on nitrate concentration in unsaturated riparian area bounded by an intermittent Mediterranean stream. Water Resour Res 39:1110CrossRefGoogle Scholar
  9. Carrigg C, Rice O, Kavanagh S, Collins G, O’Flaherty V (2007) DNA extraction method affects microbial community profiles from soils and sediment. App Microbiol Biotechnol 77(4):955–964CrossRefGoogle Scholar
  10. Cho JC, Kim SJ (2000) Increase in bacterial community diversity in subsurface aquifers receiving livestock wastewater input. Appl Environ Microbiol 66(3):956–965CrossRefGoogle Scholar
  11. Clarke KR (1993) Non-parametric multivariate analyses of changes in community structure. Aust J Ecol 18:117–143CrossRefGoogle Scholar
  12. Guo H, Li G, Zhang D, Xu Z, Lu C’a (2006) Effects of water table and fertilization management on nitrogen loading to groundwater. Agric Water Manag 82:86–98CrossRefGoogle Scholar
  13. 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):5181–5189Google Scholar
  14. Hill AR (1996) Nitrate removal in stream riparian zones. J Environ Qual 25:743–755CrossRefGoogle Scholar
  15. Hord NG, Tang Y, Bryan NS (2009) Food sources of nitrates and nitrites: the physiologic context for potential health benefits. Am J Clin Nutr 90:1–10CrossRefGoogle Scholar
  16. IPCC (2001) Good practice guidance and uncertainty management in national greenhouse gas inventories—indirect N2O emissions from agriculture. Intergovernmental Panel on Climate Change, GenevaGoogle Scholar
  17. Jahangir MMR, Johnston P, Khalil MI, Richards KG (2012a) Linking hydrogeochemistry to nitrate abundance in groundwater in agricultural settings in Ireland. J Hydrol 448:212–222Google Scholar
  18. Jahangir MMR, Johnston P, Khalil MI, Richards KG (2012b) Groundwater: a pathway of terrestrial C and N losses and indirect greenhouse gas emissions. Agric Ecosyst Environ 159:40–48Google Scholar
  19. Kana TM, Darkenjelo C, Hunt MD, Oldham JB, Bennett GE, Cornwell JC (1994) Membrane inlet mass spectrometer for rapid high-precision determination of N2, O2, and Ar in environmental water samples. Anal Chem 66:4166–4170CrossRefGoogle Scholar
  20. 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 Microb 72(9):5957–5962Google Scholar
  21. Luo J, Tillman RW, Ball PR (1999) Factors regulating denitrification in a soil under pasture. Soil Biol Biochem 31:913–927CrossRefGoogle Scholar
  22. Minamikawa K, Nishimura S, Sawamoto T, Nakajima Y, Yagi K (2010) Annual emissions of dissolved CO2, CH4 and N2O in the subsurface drainage from three cropping systems. Glob Chang Biol 16:796–809CrossRefGoogle Scholar
  23. Modin O, Fukushi K, Nakajima K, Yamamoto K (2010) Nitrate removal and biofilm characteristics in methanotrophic membrane biofilm reactors with various gas supply regimes. Water Res 44(1):85–96Google Scholar
  24. Nevison C (2000) Review of the IPCC methodology for estimating nitrous oxide emissions associated with agriltural leaching and runoff. Chemosphere-Global Change Sci 2:493–500CrossRefGoogle Scholar
  25. Nziguheba G, Smolders E (2008) Inputs of trace elements in agricultural soils via phosphate fertilizers in European countries. Sci Total Environ 390(1):53–57CrossRefGoogle Scholar
  26. Peterjohn WT, Correll DL (1984) Nutrient dynamics in an agricultural watershed: observations on the role of a riparian forest. Ecology 65(5):1466–1475CrossRefGoogle Scholar
  27. Rahn RC et al (2010) EU-Rotate_N—a European decision support system—to predict environmental and economic consequences of the management of nitrogen fertiliser in crop rotations. Eur J Hortic Sci 75:20–32Google Scholar
  28. Richardson D, Felgate G, 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 in Biotechnology 27(7):388–397Google Scholar
  29. Rodgers M., Gibbons P., Mulqueen J. (2003). Nitrate leaching on a sandy loam soil under different dairy wastewater applications. Diffuse Pollution Conference Dublin 2003 7A Groundwater 7–14.Google Scholar
  30. Santoro AE, Boehm AB, Francis CA (2006) Denitrifier community composition along a nitrate and salinity gradient in a coastal aquifer. Appl Environ Microbiol 72(3):2102–2109CrossRefGoogle Scholar
  31. Sei K, Asano K, Tateishi N, Mori K, Ike M, Fujita M (1999) Design of PCR primers and gene probes for the general detection of bacterial populationscapable of degrading aromatic compounds via catechol cleavage pathways. J Biosci Bioeng 88(5):542–550CrossRefGoogle Scholar
  32. Soares MIM (2000) Biological denitrification of groundwater. Water Air Soil Pollut 123:183–193CrossRefGoogle Scholar
  33. Spalding RF, Parrott JD (1994) Shallow groundwater denitrification. Sci Total Environ 141:17–25CrossRefGoogle Scholar
  34. Stark CH, Richards KG (2008a) The continuing challenge of agricultural nitrogen loss to the environment in the context of global change and advancing research. Dyn Soil Dyn Plant 2:1–12Google Scholar
  35. Stark CH, Richards KG (2008b) The continuing challenge of nitrogen loss to the environment: environmental consequences and mitigation strategies. Dyn Soil Dyn Plant 2:41–55Google Scholar
  36. Strebel O, Bőttcher J (1989) Solute input into groundwater from sandy soils under arable land and coniferous forest: determination of area-representative mean values of concentration. Agric Water Manag 15:265–278CrossRefGoogle Scholar
  37. Surridge BWJ, Heathwaite AL, Baird AJ (2007) The release of phosphorus to porewater and surface water from river riparian sediments. J Environ Qual 36:1534–1544CrossRefGoogle Scholar
  38. Tindall JA, Petrusak RL, McMahon C, Peter B (1995) Nitrate transport and transformation processes in unsaturated porous media. J Hydrol 169(1–4):51–94CrossRefGoogle Scholar
  39. Torrentó C, Urmeneta J, Otero N, Soler A, Viñas M, Cama J (2011) Enhanced denitrification in groundwater and sediments from a nitrate-contaminated aquifer after addition of pyrite. Chem Geol 287:90–101CrossRefGoogle Scholar
  40. USEPA (2009) Article 11—Attachment A1—proposed maximum concentration limits. US Environmental Protection Agency, WashingtonGoogle Scholar
  41. Venterea RT, Hyatt CR, Rosen CJ (2011) Fertilizer management effects on nitrate leaching and indirect nitrous oxide emissions in irrigated potato production. J Environ Qual 40:1103–1112CrossRefGoogle Scholar
  42. Von der Heide C, Boettcher J, Deurer M, Duijnisveld W, Weymann D, Well R (2010) Spatial and temporal variability of N2O in the surface groundwater; a detailed analysis from a sandy aquifer in northern Germany. Nutr Cycl Ecosyst 87:33–47CrossRefGoogle Scholar
  43. Weymann D, Well R, Flessa H, von der Heide C, Duerer M, Meyer K, Konrad C, Walther W (2008) Groundwater N2O emission factors of nitrate- contaminated aquifers as derived from denitrification progress and N2O accumulation. Biogeosci Discuss 5:1215–1226CrossRefGoogle Scholar
  44. Weymann D, Well R, von der Heide C, Böttcher J, Flessa H, Duijnisveld WHM (2009) Recovery of groundwater N2O at the soil surface and its contribution to total N2O emissions. Nutr Cycl Agroecosyst 85:299–312CrossRefGoogle Scholar
  45. World Health Organisation (WHO) (1984) Guidelines for drinking water quality. World Health Organ Geneva 2:290–292Google Scholar
  46. Yan TF, Fields MW, Wu LY, Zu YG, Tiedje JM, Zhou JZ (2003) Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater. Environ Microbiol 5:13–24CrossRefGoogle Scholar
  47. 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):670–679Google Scholar
  48. Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62(2):316–322Google Scholar
  49. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Maria Barrett
    • 1
  • Mohammad M. R. Jahangir
    • 2
  • Changsoo Lee
    • 3
    • 9
  • Cindy J. Smith
    • 4
    • 5
  • Niamh Bhreathnach
    • 1
  • Gavin Collins
    • 5
    • 6
    • 7
  • Karl G. Richards
    • 8
  • Vincent O’Flaherty
    • 1
    • 5
  1. 1.Microbial Ecology Laboratory, School of Natural SciencesNational University of Ireland Galway (NUI Galway)GalwayIreland
  2. 2.Civil, Structural & Environmental EngineeringTrinity College DublinDublin 2Ireland
  3. 3.School of Civil & Environmental EngineeringNanyang Technological UniversityNanyangSingapore
  4. 4.Marine Microbial Ecology Laboratory, School of Natural SciencesNUI GalwayGalwayIreland
  5. 5.Ryan Institute for Environmental, Marine & Energy ResearchNUI GalwayGalwayIreland
  6. 6.Microbial Ecophysiology Laboratory, School of Natural SciencesNUI GalwayGalwayIreland
  7. 7.Infrastructure and Environment, School of EngineeringUniversity of GlasgowGlasgowUK
  8. 8.Teagasc Environment Research CentreJohnstown Castle, CoWexfordIreland
  9. 9.School of Urban & Environmental EngineeringUlsan National Institute of Science & TechnologyUlsanRepublic of Korea

Personalised recommendations