Combined Use of the Acetylene Inhibition Technique and Microsensors for Quantification of Denitrification in Sediments and Biofilms

  • Niels Peter Revsbech
  • Jan Sørensen
Part of the Federation of European Microbiological Societies Symposium Series book series (FEMS, volume 56)


The introduction of the acetylene inhibition technique for quantification of denitrification (Balderston et al. 1976; Yoshinari and Knowles, 1976) supplied researchers of this reaction with a highly sensitive and relatively simple assay. Nitrous oxide is a free intermediary in the bacterial reduction of NO 3 to N2, and the reduction of N2O is inhibited by acetylene. The accumulation rate of N2O in the inhibited sample is thus a measure of the denitrification activity. Gas chromatographs equipped with ECDs (Electron Capture Detectors) can be used to quantify the evolved N2O with good accuracy and extremely high detection limits. A problem by use of gas chromatography is, however, the need to extract nitrous oxide from the sample before analysis. The procedures for extraction of sample segments may be elaborated to yield a reasonable spatial resolution of the assay (e.g., Sørensen et al, 1979), but the highest resolution by such an approach is still rather on a centimeter than on a millimeter scale while denitrifying microenvironments such as biofilms often have dimensions of less than 1 mm. The recent introduction of a microsensor for simultaneous detection of O2 and N2O has now made it possible to determine the activity within denitrifying microenvironments with a spatial resolution of less than 0.1 mm.


Apparent Diffusion Coefficient Sediment Core Overlie Water Denitrification Activity Dissimilatory Reduction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aller, R.C., 1980, Quantifying solute distributions in the bioturbated zone of marine sediments by defining an average microenvironment, Geochim. Cosmochim. Acta, 44: 1955.CrossRefGoogle Scholar
  2. Andersen, T.K., Jensen, M.H., and J. Sorensen, 1984, Diurnal variation of nitrogen cycling in coastal, marine sediments. I. Denitrification, Mar. Biol., 83:171.CrossRefGoogle Scholar
  3. Broecker, W.S., and Peng, T.-H., 1974, Gas exchange rates between air and sea, Tellus, 26: 21.CrossRefGoogle Scholar
  4. Balderston, W.L., Sherr, B., and Payne, W.J., 1976, Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus, Appl. Environ. Microbiol., 31: 504.Google Scholar
  5. Berner, R.A., 1980, “Early diagenesis. A theoretical approach,” Princeton University Press, Princeton.Google Scholar
  6. Broecker, W.S., and Peng, T.-H., 1974, Gas exchange rates between air and sea, Tellus, 26: 21.CrossRefGoogle Scholar
  7. Capone, D.G., and Bautista, M., 1985, Direct evidence for a groundwater source for nitrate in nearshore marine sediments, Nature, 313: 143.CrossRefGoogle Scholar
  8. Chang, Q., and Mayerhof, E., 1986, Membrane-dialyzer injection loop for enhancing the selectivity of anion-responsive liquid-membrane electrodes in flow systems, Anal. Chim. Acta., 186: 81.CrossRefGoogle Scholar
  9. Christensen, P.B., Nielsen, L.P., Revsbech, N.P., and Sorensen, J., 1989, Microzonation of denitrification activity in stream sediments as studied with a combined oxygen and nitrius oxide microsensor, Appl. Environ. Microbiol., 55: 1234.PubMedGoogle Scholar
  10. Christensen, P.B., and Sorensen, J., 1986, Temporal variation of denitrification activity in plant covered littoral sediment (Lake Hampen, Denmark). Appl. Environ. Microbiol., 51: 1174.PubMedGoogle Scholar
  11. Dalsgaard, T., 1988, “Denitrification in biofilms,” M.Sc. thesis, University of Aarhus, Aarhus.Google Scholar
  12. de Beer, D., and Sweerts, J.-P.R.A., 1989, Measurement of nitrate gradients with an ion-selective microelectrode. Anal. Chim. Acta, 219: 351.CrossRefGoogle Scholar
  13. Gust, G., Booij, K., Helder, W., and Sundby, B., 1987, On the velocity sensitivity (stirring effect) of polarographic oxygen microelectrodes, Neth. J. Sea Res., 21: 255.CrossRefGoogle Scholar
  14. Kaspar, H.F., Tiedje, J.M., and Firestone, R.B., 1981, Denitrification and dissimilatory nitrate reduction to ammonium by digested sludge, Can. J. Microbiol., 38: 486.Google Scholar
  15. Koike, I., and Sorensen, J., 1988, Nitrate reduction and denitrification in marine sediments, in: “Nitrogen cycling in coastal marine environments,” T.H. Blackburn and J. Sorensen, eds., SCOPE 33, John Wiley & Sons, Chichester.Google Scholar
  16. Li, Y.-H., and Gregory, S., 1974, Diffusion of ions in sea water and in deep sea sediments, Geochim. Cosmochim. Acta., 38: 703.CrossRefGoogle Scholar
  17. Nishio, T., 1982, “Nitrogen cycling in coastal and estuarine sediments with special reference to nitrate reduction, denitrification and nitrification,” Doctoral dissertation, University of Tokyo, Tokyo.Google Scholar
  18. Oremland, R.S., Umberger, C., Culbertson, C.W., and Smith, R.L., 1984, Denitrification in San Fransisco intertidal sediments, Appl. Environ. Microbiol., 47: 1106.PubMedGoogle Scholar
  19. Revsbech, N.P., 1983, In situ measurement of oxygen profiles of sediments by use of oxygen microelectrodes, in: “Polarographic oxygen sensors: Aquatic and physiological applications,” E. Gnaiger and H. Forster, eds., Springer, Heidelberg.Google Scholar
  20. Revsbech, N.P., 1989a, An oxygen microsensor with a guard cathode, Limnol. Oceanogr., 34: 474.CrossRefGoogle Scholar
  21. Revsbech, N.P., 1989b, Diffusion characteristics of microbial communities determined by use of oxygen microsensors. J. Microbiol. Meth., 9: 111.CrossRefGoogle Scholar
  22. Revsbech, N.P., Christensen, P.B., Nielsen, L.P., and Sorensen, J., 1988, A combined oxygen and nitrous oxide microsensor for denitrification studies. Appl. Environ. Microbiol., 54: 2245.PubMedGoogle Scholar
  23. Revsbech, N.P., Christensen, P.B., Nielsen, L.P., and J. Sorensen, 1989, Denitrification in a trickling filter biofilm studied by a microsensor for oxygen and nitrous oxide, Water Res., 23: 867.CrossRefGoogle Scholar
  24. Revsbech, N.P., and Jorgensen, B.B., 1986, Microelectrodes: Their use in microbial ecology, in: “Advances in Microbial Ecology, vol. 9,” K.C. Marshall, ed., Plenum, New York.Google Scholar
  25. Revsbech, N.P., Madsen, B., and Jorgensen, B.B., 1986, Oxygen production and consumption in sediments determined at high spatial resolution by computer simulation of oxygen microelectrode data, Limnol. Oceanogr., 31: 293.CrossRefGoogle Scholar
  26. Riemer, M., and Harremoes, P., 1978, Multi-component diffusion in denitrifying biofilms, Prog. Wat. Res., 10: 149.Google Scholar
  27. Sextone, A.J., Revsbech, N.P., Parkin, T.B., and Tiedje, J.M., 1985, Direct measurement of oxygen profiles and denitrification rates in soil aggregates, Soil. Sci. Soc. Am. J., 49: 645.CrossRefGoogle Scholar
  28. Slater, J.M., and Capone, D.G., 1989, Nitrate requirement for acetylene inhibition of nitrous oxide reduction in marine sediments, Microb. Ecol., 17: 143.CrossRefGoogle Scholar
  29. Sørensen, J, 1978, Denitrification rates in a marine sediment as measured the acetylene inhibition technique, Appl. Environ. Microbiol., 36: 139.PubMedGoogle Scholar
  30. Sørensen, J., Jorgensen, B.B., and Revsbech, N.P., 1979, A comparison of oxygen, nitrate, and sulfate respiration in coastal marine sediments, Microb. Ecol., 5: 105.CrossRefGoogle Scholar
  31. Sørensen, J., Rasmussen, L.K., and Koike, I., 1987, Micromolar sulfide concentration alleviate acetylene blockage of nitrous oxide reduction by denitrifying Pseudomonas fluorescens. Can. J. Microbiol., 33: 1001.CrossRefGoogle Scholar
  32. Sweerts, J.-P.R.A., and de Beer, D., 1989, Microelectrode measurements of nitrate gradients in the littoral and profundal sediments of a meso-eutrophic lake (Lake Vechten, The Netherlands), Appl. Environ. Microbiol., 55: 754.PubMedGoogle Scholar
  33. Tam, T.-Y., and Knowles, R., 1979. Effects of sulfide and acetylene on nitrous oxide reduction by soil and by Pseudomonas aeruginosa, Can. J. Microbiol., 25: 1133.CrossRefGoogle Scholar
  34. Thomas, R.C., 1978, “Ion-sensitive intracellular microelectrodes, how to make and use them”. Academic Press, London.Google Scholar
  35. Yoshinari, T., and Knowles, R., 1976, Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochem. Biophys. Res. Commun., 69: 705.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • Niels Peter Revsbech
    • 1
  • Jan Sørensen
    • 2
  1. 1.Department of Ecology and GeneticsUniversity of AarhusAarhus CDenmark
  2. 2.Department of Ecology and Molecular Biology Section of MicrobiologyRoyal Veterinary and Agricultural UniversityFrederiksberg CDenmark

Personalised recommendations