Applied Microbiology and Biotechnology

, Volume 89, Issue 1, pp 189–200 | Cite as

Field application of nitrogen and phenylacetylene to mitigate greenhouse gas emissions from landfill cover soils: effects on microbial community structure

  • Jeongdae Im
  • Sung-Woo Lee
  • Levente Bodrossy
  • Michael J. Barcelona
  • Jeremy D. Semrau
Environmental Biotechnology


Landfills are large sources of CH4, but a considerable amount of CH4 can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N2O production. To examine the effects of nitrogen and a selective inhibitor on CH4 oxidation and N2O production in situ, 0.5 M of NH4Cl and 0.25 M of KNO3, with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N2O production but had no effect on CH4 oxidation. The addition of phenylacetylene stimulated CH4 oxidation while reducing N2O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N2O production by selectively inhibiting AOAs and/or type II methanotrophs.


Methane Nitrous oxide Methanotrophs Ammonia-oxidizing archaea 



The authors would like to acknowledge the field sampling and analyses contributions of Peter Stuurwold and William Lizik (Western Michigan University). The provision of an archaeal amoA clone from Dr. Craig S. Criddle is also gratefully acknowledged. This project was supported by the Department of Energy (DE-FC26-05NT42431).

Supplementary material

253_2010_2811_Fig1_ESM.gif (164 kb)
Supplementary Figure S1

Rarefaction analyses of amoA genes from ammonia-oxidizing archaea. OTUs were defined as those with ≥97% nucleotide sequence identity. Error bars represent 95% confidence limits. (GIF 163 kb)

253_2010_2811_MOESM1_ESM.eps (373 kb)
High resolution image file (EPS 373 KB)


  1. Anderson IC, Levine JS (1986) Relative rates of nitric oxide and nitrous oxide production by nitrifiers, denitrifiers, and nitrate respirers. Appl Environ Microbiol 51:938–945Google Scholar
  2. Arp DJ, Stein LY (2003) Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit Rev Biochem Mol Biol 38:471–495CrossRefGoogle Scholar
  3. Barlaz MA, Green RB, Chanton JP, Goldsmith CD, Hater GR (2004) Evaluation of a biologically active cover for mitigation of landfill gas emissions. Environ Sci Technol 38:4891–4899CrossRefGoogle Scholar
  4. Berge N, Reinhart D, Townsend T (2005) The fate of nitrogen in bioreactor landfills. Environ Sci Technol 35:365–399Google Scholar
  5. Beman JM, Popp BN, Francis CA (2008) Molecular and biogeochemical evidence for ammonia oxidation by marine Crenarchaeota in the Gulf of California. ISME J 2:429–441CrossRefGoogle Scholar
  6. Bodelier PLE, Roslev P, Henckel T, Frenzel P (2000) Stimulation by ammonium-based fertilizers of methane oxidation in soil around rice roots. Nature 403:421–424CrossRefGoogle Scholar
  7. Bodrossy L, Stralis-Pavese N, Murrell JC, Radajewski S, Weilharter A, Sessitsch A (2003) Development and validation of a diagnostic microbial microarray for methanotrophs. Environ Microbiol 5:566–582CrossRefGoogle Scholar
  8. Bogner JE, Spokas KA, Burton EA (1997) Kinetics of methane oxidation in a landfill cover soil: temporal variations, a whole-landfill oxidation experiment, and modeling of net CH4 emissions. Environ Sci Technol 31:2504–2514CrossRefGoogle Scholar
  9. Borjesson G, Svensson BH (1997) Nitrous oxide emissions from landfill cover soils in Sweden. Tellus B 49:357–363CrossRefGoogle Scholar
  10. Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics 4:29CrossRefGoogle Scholar
  11. Casciotti KL, Ward BB (2005) Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria. FEMS Microbiol Ecol 52:197–205CrossRefGoogle Scholar
  12. Cebron A, Bodrossy L, Chen Y, Singer AC, Thompson IP, Prosser JI, Murrell JC (2007) Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing. FEMS Microbiol Ecol 62:12–23CrossRefGoogle Scholar
  13. Costello AM, Lidstrom ME (1999) Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. Appl Environ Microbiol 65:5066–5074Google Scholar
  14. Crutzen PJ (1991) Methane’s sinks and sources. Nature 350:380–381CrossRefGoogle Scholar
  15. Firestone MK, Firestone RB, Tiedje JM (1980) Nitrous oxide production from soils: factors controlling its biological production. Science 208:749–751CrossRefGoogle Scholar
  16. Francis CA, Roberts KJ, Beman JM, Santoro AE, Oakley BB (2005) Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proc Natl Acad Sci USA 102:14683–14688CrossRefGoogle Scholar
  17. Graham DW, Chaudhary JA, Hanson RS, Arnold RG (1993) Factors affecting competition between Type I and Type II methanotrophs in two-organism, continuous-flow reactors. Microb Ecol 25:1–17CrossRefGoogle Scholar
  18. Halet D, Boon N, Verstraete W (2006) Community dynamics of methanotrophic bacteria during composting of organic matter. J Biosci Bioeng 101:297–302CrossRefGoogle Scholar
  19. Hallin S, Lindgren PE (1999) PCR detection of genes encoding nitrite reductase in denitrifying bacteria. Appl Environ Microbiol 65:1652–1657Google Scholar
  20. Han JI, Semrau JD (2004) Quantification of gene expression in methanotrophs by competitive reverse transcription-polymerase chain reaction. Environ Microbiol 6:388–399CrossRefGoogle Scholar
  21. Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, Daims H, Wagner M (2008) A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. Proc Natl Acad Sci 105:2134–2139CrossRefGoogle Scholar
  22. Hutchens E, Radajewski S, Dumont MG, McDonald IR, Murrell JC (2004) Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing. Environ Microbiol 6:111–120CrossRefGoogle Scholar
  23. IPCC (2007) Climate Change 2007: synthesis report. Cambridge University Press, CambridgeGoogle Scholar
  24. Jiang QQ, Bakken LR (1999) Nitrous oxide production and methane oxidation by different ammonia oxidizing bacteria. Appl Environ Microbiol 65:2679–2684Google Scholar
  25. Kester RA, de Boer W, Laanbroek HJ (1996) Short exposure to acetylene to distinguish between nitrifier and denitrifier nitrous oxide production in soil and sediment samples. FEMS Microbiol Ecol 20:111–120Google Scholar
  26. Kightley D, Nedwell D, Cooper M (1995) Capacity for methane oxidation in landfill cover soils measured in laboratory-scale soil microcosms. Appl Environ Microbiol 61:592–601Google Scholar
  27. Könneke M, Bernhard AE, Torre JR, Walker JB, Waterbury JB, Stahl DA (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437:543–546CrossRefGoogle Scholar
  28. Kreader CA (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 62:1102–1106Google Scholar
  29. Leinenger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442:806–809CrossRefGoogle Scholar
  30. Lee S-W, Keeney DR, Lim D-H, DiSpirito AA, Semrau JD (2006) Mixed pollutant degradation by Methylosinus trichosporium OB3b expressing either soluble or particulate methane monooxygenase: can the tortoise beat the hare? Appl Environ Microbiol 72: 7503–7509CrossRefGoogle Scholar
  31. Lee S-W, Im J, DiSpirito A, Bodrossy L, Barcelona M, Semrau J (2009) Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers. Appl Microbiol Biotechnol 85:389–403CrossRefGoogle Scholar
  32. Lontoh S, DiSpirito AA, Krema CL, Whittaker MR, Hooper AB, Semrau JD (2000) Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monooxygenase by phenylacetylene. Environ Microbiol 2:485–494CrossRefGoogle Scholar
  33. Majumdar D (2003) Methane and nitrous oxide emission from irrigated rice fields: proposed mitigation strategies. Curr Sci 84:1317–1326Google Scholar
  34. Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA (2009) Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461:976–979CrossRefGoogle Scholar
  35. McBride MB, Richards BK, Steenhuis T (2004) Bioavailability and crop uptake of trace elements in soil columns amended with sewage sludge products. Plant Soil 262:71–84CrossRefGoogle Scholar
  36. Milke MW (1998) Computer simulation to evaluate the economics of landfill gas recovery. Water Sci Technol 38:201–208Google Scholar
  37. Mohanty SR, Bodelier PLE, Floris V, Conrad R (2006) Differential effects of nitrogenous fertilizers on methane-consuming microbes in rice field and forest soils. Appl Environ Microbiol 72:1346–1354CrossRefGoogle Scholar
  38. Nicol GW, Schleper C (2006) Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle? Trends Microbiol 14:207–212CrossRefGoogle Scholar
  39. Nicol GW, Glover LA, Prosser JI (2003) The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil. Environ Microbiol 5:152–162CrossRefGoogle Scholar
  40. Nicol GW, Tscherko D, Embley TM, Prosser JI (2005) Primary succession of soil Crenarchaeota across a receding glacier forland. Environ Microbiol 7:337–347CrossRefGoogle Scholar
  41. Ochsenreiter T, Selezi D, Quaiser A, Bonch-Osmolovskaya L, Schleper C (2003) Diversity and abundance of Crenarchaeota in terrestrial habitats studied by 16S RNA surveys and real time PCR. Environ Microbiol 5:787–797CrossRefGoogle Scholar
  42. Okana Y, Hristova KR, Leutenegger CM, Jackson LE, Denison RF, Gebreyesus B, Lebauer D, Scow KM (2004) Application of real-time PCR to study effects of ammonium on population size of ammonia-oxidizing bacteria in soil. Appl Environ Microbiol 70:1008–1016CrossRefGoogle Scholar
  43. Offre P, Prosser JI, Nicol GW (2009) Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene. FEMS Microbiol Ecol 70:99–108CrossRefGoogle Scholar
  44. Park S, Lee CH, Ryu CR, Sung KJ (2009) Biofiltration for reducing methane emissions from modern sanitary landfills at the low methane generation stage. Water Air Soil Pollut 196:19–27CrossRefGoogle Scholar
  45. Philopoulos A, Ruck J, McCartney D, Felske C (2009) A laboratory-scale comparison of compost and sand-compost-perlite as methane-oxidizing biofilter media. Waste Manage Res 27:138–146CrossRefGoogle Scholar
  46. Prosser JI, Nicol GW (2008) Relative contributions of archaea and bacteria to aerobic ammonia oxidation in the environment. Environ Microbiol 10:2931–2941CrossRefGoogle Scholar
  47. Reinhart DR, McCreanor PT, Townsend T (2002) The bioreactor landfill: its status and future. Waste Manage Res 20:172–186CrossRefGoogle Scholar
  48. Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker, molecular fine scale analysis of natural ammonia oxidizing populations. Appl Environ Microbiol 63:4704–4712Google Scholar
  49. Semrau JD, DiSpirito AA, Yoon S (2010) Methanotrophs and copper. FEMS Microbiol Rev 34:496–531Google Scholar
  50. Simon HM, Jahn CE, Bergerud LT, Sliwinski MK, Weimer PJ, Willis DK, Goodman RM (2005) Cultivation of mesophilic crenarchaeotes in enrichment cultures from plant roots. Appl Environ Microbiol 75:4751–4760CrossRefGoogle Scholar
  51. Soil Survey Division Staff (1993) Soil survey manual. US Department of Agriculture Handbook 18. US Government Printing Office, Washington, DCGoogle Scholar
  52. Stralis-Pavese N, Sessitsch A, Weilharter A, Reichenauer T, Riesing J, Js C, Murrell JC, Bodrossy L (2004) Optimization of diagnostic microarray for application in analysing landfill methanotroph communities under different plant covers. Environ Microbiol 6:347–363CrossRefGoogle Scholar
  53. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefGoogle Scholar
  54. Tourna M, Freitag TE, Nicol GW, Prosser JI (2008) Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms. Environ Microbiol 10:1357–1364CrossRefGoogle Scholar
  55. Webster G, Embley TM, Freitag TE, Smith Z, Prosser JI (2005) Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils. Environ Microbiol 7:676–684CrossRefGoogle Scholar
  56. Whalen SC, Reeburgh WS, Sandbeck KA (1990) Rapid methane oxidation in a landfill cover soil. Appl Environ Microbiol 56:3405–3411Google Scholar
  57. Whittenbury R, Phillips KC, Wilkinson JG (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61:205–218Google Scholar
  58. Wuchter C, Abbas B, Coolen MJL, Herfort L, van Bleijswijk J, Timmers P, Strous M, Teira E, Herndl GJ, Middelburg JJ, Schouten S, Damste JSS (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci USA 103:12317–12322CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Jeongdae Im
    • 1
  • Sung-Woo Lee
    • 1
    • 4
  • Levente Bodrossy
    • 2
    • 5
  • Michael J. Barcelona
    • 3
  • Jeremy D. Semrau
    • 1
  1. 1.Department of Civil and Environmental EngineeringThe University of MichiganAnn ArborUSA
  2. 2.Department of BiotechnologyARC Seibersdorf Research GmbHSeibersdorfAustria
  3. 3.Department of ChemistryWestern Michigan UniversityKalamazooUSA
  4. 4.Oregon Graduate Institute, Department of Environmental & Biomolecular SystemsOregon Health and Sciences UniversityBeavertonUSA
  5. 5.CSIRO Marine and Atmospheric ResearchHobartAustralia

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