Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Mar;61(3):1110–1115. doi: 10.1128/aem.61.3.1110-1115.1995

Criteria and Methodology for Identifying Respiratory Denitrifiers

I Mahne, J M Tiedje
PMCID: PMC1388392  PMID: 16534960

Abstract

Respiratory denitrification is not always adequately established when bacteria are characterized. We have tested a simple method that allows one to evaluate whether the two necessary criteria to claim denitrification have been met, namely, that N(inf2) or N(inf2)O is produced from nitrate or nitrite and that this reduction is coupled to a growth yield increase. Microorganisms were cultured in sealed tubes under a helium headspace and in the presence of 0, 2, 4, 7, and 10 mM nitrate or nitrite. After growth had ceased, N(inf2) and N(inf2)O were quantified by gas chromatography and the final protein concentration was measured. Net protein production was linearly related to nitrate concentration for all denitrifiers tested and ranged from 2 to 6 g of protein per mol of electron equivalent reduced. Nitrogen recovery as N(inf2) plus N(inf2)O from nitrate and nitrite transformed exceeded 80% for all denitrifiers. We also suggest that a rate of N gas production of >10 (mu)mol/min/g of protein can be used as an additional characteristic definitive of denitrification since this process produces gas more rapidly than other processes. These characteristics were established after evaluation of a variety of well-characterized respiratory denitrifiers and other N(inf2)O-producing nitrate reducers. Several poorly characterized denitrifiers were also tested and confirmed as respiratory denitrifiers, including Aquaspirillum itersonii, Aquaspirillum fasciculus, Bacillus azotoformans, and Corynebacterium nephridii. These criteria distinguished respiratory denitrifiers from other groups that reduce nitrate or produce N(inf2)O. Furthermore, they correctly identified respiratory denitrification in weak denitrifiers, a group in which the existence of this process may be overlooked.

Full Text

The Full Text of this article is available as a PDF (270.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Betlach M. R. Evolution of bacterial denitrification and denitrifier diversity. Antonie Van Leeuwenhoek. 1982;48(6):585–607. doi: 10.1007/BF00399543. [DOI] [PubMed] [Google Scholar]
  2. Bleakley B. H., Tiedje J. M. Nitrous oxide production by organisms other than nitrifiers or denitrifiers. Appl Environ Microbiol. 1982 Dec;44(6):1342–1348. doi: 10.1128/aem.44.6.1342-1348.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlson C. A., Ingraham J. L. Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Appl Environ Microbiol. 1983 Apr;45(4):1247–1253. doi: 10.1128/aem.45.4.1247-1253.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caskey W. H., Tiedje J. M. The reduction of nitrate to ammonium by a Clostridium sp. isolated from soil. J Gen Microbiol. 1980 Jul;119(1):217–223. doi: 10.1099/00221287-119-1-217. [DOI] [PubMed] [Google Scholar]
  5. Coyne M. S., Arunakumari A., Averill B. A., Tiedje J. M. Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria. Appl Environ Microbiol. 1989 Nov;55(11):2924–2931. doi: 10.1128/aem.55.11.2924-2931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eskew D. L., Focht D. D., Ting I. P. Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum. Appl Environ Microbiol. 1977 Nov;34(5):582–585. doi: 10.1128/aem.34.5.582-585.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gamble T. N., Betlach M. R., Tiedje J. M. Numerically dominant denitrifying bacteria from world soils. Appl Environ Microbiol. 1977 Apr;33(4):926–939. doi: 10.1128/aem.33.4.926-939.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hochstein L. I., Tomlinson G. A. The enzymes associated with denitrification. Annu Rev Microbiol. 1988;42:231–261. doi: 10.1146/annurev.mi.42.100188.001311. [DOI] [PubMed] [Google Scholar]
  9. Hollocher T. C. The pathway of nitrogen and reductive enzymes of denitrification. Antonie Van Leeuwenhoek. 1982;48(6):531–544. doi: 10.1007/BF00399539. [DOI] [PubMed] [Google Scholar]
  10. Koike I., Hattori A. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite- and oxide-limited conditions. J Gen Microbiol. 1975 May;88(1):11–19. doi: 10.1099/00221287-88-1-11. [DOI] [PubMed] [Google Scholar]
  11. Koike I., Hattori A. Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions. J Gen Microbiol. 1975 May;88(1):1–10. doi: 10.1099/00221287-88-1-1. [DOI] [PubMed] [Google Scholar]
  12. Patriquin D. G., Knowles R. Denitrifying bacteria in some shallow-water marine sediments: enumeration and gas production. Can J Microbiol. 1974 Jul;20(7):1037–1041. doi: 10.1139/m74-160. [DOI] [PubMed] [Google Scholar]
  13. Rehr B., Klemme J. H. Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria. Antonie Van Leeuwenhoek. 1989 Nov;56(4):311–321. doi: 10.1007/BF00443745. [DOI] [PubMed] [Google Scholar]
  14. Shoun H., Tanimoto T. Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction. J Biol Chem. 1991 Jun 15;266(17):11078–11082. [PubMed] [Google Scholar]
  15. Smith G. B., Tiedje J. M. Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria. Appl Environ Microbiol. 1992 Jan;58(1):376–384. doi: 10.1128/aem.58.1.376-384.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Thauer R. K., Jungermann K., Decker K. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev. 1977 Mar;41(1):100–180. doi: 10.1128/br.41.1.100-180.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ye R. W., Averill B. A., Tiedje J. M. Denitrification: production and consumption of nitric oxide. Appl Environ Microbiol. 1994 Apr;60(4):1053–1058. doi: 10.1128/aem.60.4.1053-1058.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ye R. W., Fries M. R., Bezborodnikov S. G., Averill B. A., Tiedje J. M. Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers. Appl Environ Microbiol. 1993 Jan;59(1):250–254. doi: 10.1128/aem.59.1.250-254.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Zumft W. G. The biological role of nitric oxide in bacteria. Arch Microbiol. 1993;160(4):253–264. doi: 10.1007/BF00292074. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES