Antonie van Leeuwenhoek

, Volume 48, Issue 6, pp 585–607 | Cite as

Evolution of bacterial denitrification and denitrifier diversity

  • Michael R. Betlach
Denitrification: Recent Advances and Future Directions


Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms. Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria. Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true.

Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group. However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification.

Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms. Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety. Both types are distinct from other nitrate reductases.

Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny. Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms. However, denitrification is restricted to genera within the purple photosynthetic group. Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes.

Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today. Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time.


Nitrate Reductase Nitrate Reduction Nitrite Reductase Taxonomic Identification Aerobic Respiration 
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.


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  1. Abraham, P. R., Boxer, D. H., Graham, A., Tucker, A. D., Van 't Riet, J. and Wientjes, F. B. 1981. Immunochemical and structural comparison of the respiratory nitrate reductase from Escherichia coli and Klebsiella aerogenes. — FEMS Microbiol. Lett. 10: 95–100.Google Scholar
  2. Alef, K. and Klemme, J.-H. 1979. Assimilatory nitrate reductase of Rhodopseudomonas capsulata AD2: A molybdo-hemeprotein. — Z. Naturforsch. 34C: 33–37.Google Scholar
  3. Alefounder, P. R., McCarthy, J. E. G. and Ferguson, S. J. 1981. The basis of the control of nitrate reduction by oxygen in Paracoccus denitrificans. — FEMS Microbiol. Lett. 12: 321–326.Google Scholar
  4. Averill, B. A. and Tiedje, J. M. 1982. The chemical mechanism of microbial denitrification. —FEBS Lett. 138: 8–12.Google Scholar
  5. Betlach, M. R. and Hochstein, L. I. 1982. Derepression of nitrate reductase in denitrifiers grown under oxygen limitation in chemostats. — Paper N65 in Abstr. Annu. Meet. Am. Soc. Microbiol., p. 188.Google Scholar
  6. Betlach, M. R. and Tiedje, J. M. 1981. Kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification. — Appl. Environ. Microbiol. 42: 1074–1084.Google Scholar
  7. Broda, E. 1975. The history of inorganic nitrogen in the biosphere. — J. Mol. Evol. 7: 87–100.Google Scholar
  8. Broda, E. and Peschek, G. A. 1979. Did respiration or photosynthesis come first?. — J. Theor. Biol. 81: 201–212.Google Scholar
  9. Buchanan, R. E. and Gibbons, N. E. 1974. Bergey's manual of determinative bacteriology. Eighth Edition. — Williams and Wilkins Co. Baltimore.Google Scholar
  10. Calder, K., Burke, K. A. and Lascelles, J. 1980. Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans — Arch. Microbiol. 126: 149–153.Google Scholar
  11. Caldwell, D. E., Caldwell, S. J. and Laycock, J. P. 1976. Thermothrix thioparus gen. et sp. nov., a facultatively anaerobic facultative chemolithotroph living at neutral pH and high temperature. — Can. J. Microbiol. 22: 1509–1517.Google Scholar
  12. Carlson, C. A. 1982. The physiological genetics of denitrifying bacteria — Antonie van Leeuwenhoek 48: 555–567.Google Scholar
  13. Chameides, W. L. and Walker, J. C. G. 1981. Rates of fixation by lightning of carbon and nitrogen in possible primitive atmospheres. — Origins Life 11: 291–302.Google Scholar
  14. Cole, J. A. and Brown, C. M. 1980. Nitrite reduction to ammonia by fermentative bacteria: a short circuit in the biological nitrogen cycle. — FEMS Microbiol. Lett. 7: 65–72.Google Scholar
  15. Coleman, K. J., Cornish-Bowden, A. and Cole, J. A. 1978. Purification and properties of nitrite reductase from Escherichia coli K 12. — Biochem. J. 175: 483–493.Google Scholar
  16. Daniel, R. M., Smith, I. M., Phillip, J. A. D., Radcliffe, H. D., Drozd, J. W. and Bull, A. T. 1980. Anaerobic growth and denitrification by Rhizobium japonicum and other rhizobia. —J. Gen. Microbiol. 120: 517–521.Google Scholar
  17. Des Marais, D. J. 1980. The organic geochemical record in ancient sediments and the early evolution of life-a short summary. p. 19–29. In H. O. Halvorson and K. E.Van Holde (eds). The origins of life and evolution, MBL Lectures in Biology, Vol. 1. — Alan R. Liss, Inc. New York.Google Scholar
  18. Downey, R. J., Kiszkiss, D. F., and Nuner, J. H. 1969. Influence of oxygen on development of nitrate respiration in Bacillus stearothermophilus. — J. Bacteriol. 98: 1056–1062.Google Scholar
  19. Egami, F. 1973. A comment to the concept on the role of nitrate fermentation and nitrate respiration in an evolutionary pathway of energy metabolism. — Z. Allg. Mikrobiol. 13: 177–181.Google Scholar
  20. Focht, D. D. and Joseph, H. 1974. Degradation of 1,1-diphenyl-ethylene by mixed cultures. —Can. J. Microbiol. 20: 631–635.Google Scholar
  21. Forget, P. 1971. Les nitrate-réductases bactériennes. Solubilisation, purification et propriétés de l'enzyme de Micrococcus denitrificans. — Eur. J. Biochem. 18: 442–450.Google Scholar
  22. Fox, G. E., Pechman, K. R. and Woese, C. R. 1977. Comparative cataloguing of 16S ribosomal ribonucleic acid; molecular approach to prokaryote systematics. — Int. J. Syst. Bacteriol. 27: 44–57.Google Scholar
  23. Fox, G. E., Stackerbrandt, E., Hespell, R. B., Bigson, J., Maniloff, J., Dyer, T. A., Wolfe, R. S., Balch, W. E., Tanner, R. S., Magrum, L. J., Zablen, L. B., Blakemore, R., Bupta, R., Bonen, L., Lewis, B. J., Stahl, D. A., Luehrsen, K. R., Chen, K. N., and Woese, C. R. 1980. The phylogeny of prokaryotes. — Science 209: 457–463.Google Scholar
  24. Garcia, J.-L. 1977. Étude de la dénitrification chez une bactérie thermophile sporulée. — Ann. Microbiol. (Inst. Pasteur) 128A: 447–458.Google Scholar
  25. Garcia, J.-L., Roussos, S. et Bensoussan 1981, Étude taxonomique de bactéries dénitrifiantes isolées sur benzoate dans des sols de riziéres du Sénégal. — Cah. O.R.S.T.O.M. Ser. Biol. 43: 13–25.Google Scholar
  26. Gest, H. 1980. The evolution of biological energy-transducing systems. — FEMS Microbiol. Lett. 7: 73–77.Google Scholar
  27. Grant, M. A. and Payne, W. J. 1981. Denitrification by strains of Neisseria, Kingella, and Chromobacterium. — Int. J. Syst. Bacteriol. 31: 276–279.Google Scholar
  28. Greenberg, E. P. and Becker, G. E. 1977. Nitrous oxide as end product of denitrification by strains of fluorescent pseudomonads — Can. J. Microbiol. 23: 903–907.Google Scholar
  29. Gribbon, J. 1982. Carbon dioxide, ammonia and life. — New Scientist 94: 413–416.Google Scholar
  30. Gudat, J. C., Singh, J. and Wharton, D. C. 1973. Cytochrome oxidase from Pseudomonas aeruginosa. I. Purification and some properties. — Biochim. Biophys. Acta 292: 376–390.Google Scholar
  31. Hall, J. B. 1973. The occurrence of nitrate on the early earth and its role in the evolution of the prokaryotes. — Space Life Sci. 4: 204–213.Google Scholar
  32. Hall, J. B. 1978. Nitrate-reducing bacteria. p. 296–298. In D. Schlessinger (ed.), Microbiliogy-1978. — American Society for Microbiology. Washington, D.C.Google Scholar
  33. Hart, L. T., Larson, A. D. and McCleskey, C. S. 1965. Denitrification by Corynebacterium nephridii. — J. Bacteriol. 89: 1104–1108.Google Scholar
  34. Hasan, S. M. and Hall, J. B. 1975. The physiological function of nitrate reduction in Clostridium perfringens. — J. Gen. Microbiol. 87: 120–128.Google Scholar
  35. Hattori, A. and Uesugi, I. 1968. Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrica. — Plant Cell Physiol. 9: 689–699.Google Scholar
  36. Hendrie, M. S., Holding, A. J. and Shewan, J. M. 1974. Emended descriptions of the genus Alcaligenes and of Alcaligenes faecalis and proposal that the generic name Achromobacter be rejected: status of the named species of Alcaligenes and Achromobacter. — Int. J. Syst. Bacteriol. 24: 534–550.Google Scholar
  37. Hollocher, T. C. 1982. The pathway of nitrogen and reductive enzymes of denitrification. — Antonie van Leeuwenhoek 48: 531–544.Google Scholar
  38. Holloway, B. W. 1979. Plasmids that mobilize bacterial chromosome. — Plasmid 2: 1–19.Google Scholar
  39. Iwasaki, H. and Matsubara, T. 1971. Cytochrome c-557 (551) and cytochrome cd of Alcaligenes faecalis. — J. Biochem. (Tokyo) 69: 847–857.Google Scholar
  40. Iwasaki, H. and Matsubara, T. 1972. A nitrite reductase from Achromobacter cycloclastes. —J. Biochem. (Tokyo) 71: 645–652.Google Scholar
  41. Iwasaki, H., Shidara, S., Suzuki, H. and Mori, T. 1963. Studies on denitrification. VII. Further purification and properties of denitrifying enzyme. — J. Biochem. (Tokyo) 53: 299–303.Google Scholar
  42. Jeter, R. M. and Ingraham, J. L. 1981. The denitrifying prokaryotes. p. 913–925. In M. P. Starr, H. Stolp, H. G. Truper, A. Ballows, and H. G. Schlegel (eds), The prokaryotes. — Springer Verlag, New York.Google Scholar
  43. Kakutani, T., Watanabe, H., Arima, K. and Beppu, T. 1981. Purification and properties of copper-containing nitrite reductase from a denitrifying bacterium, Alcaligenes faecalis strain S-6. — J. Biochem. (Tokyo) 89: 453–461.Google Scholar
  44. Kaneko, M. and Ishimoto, M. 1978. A study on nitrate reductase from Propionibacterium acidipropionici. — J. Biochem. (Tokyo) 83: 191–200.Google Scholar
  45. Knowles, R. 1981. Denitrification. p. 323–369. In E. A. Paul and J. N. Ladd (eds), Soil biochemistry, Vol. 5. — Marcel Dekker, Inc. New York.Google Scholar
  46. Krieg, N. R. 1976. Biology of the chemoheterotrophic spirilla. — Bacteriol. Rev. 40: 55–115.Google Scholar
  47. Lam, Y. and Nicholas, D. J. D. 1969. A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans. — Biochim. Biophys. Acta 180: 459–472.Google Scholar
  48. Liu, M.-C. and Peck, H. D.Jr. 1981. The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. — J. Biol. Chem. 256: 13159–13164.Google Scholar
  49. MacKay, R. M., Zablen, L. B., Woese, C. R. and Doolittle, W. F. 1979. Homologies in processing and sequence between the 23S ribosomal ribonucleic acids of Paracoccus denitrificans and Rhodopseudomonas sphaeroides. — Arch. Microbiol. 123: 165–172.Google Scholar
  50. Mendez, J. M. and Vega, J. M. 1981. Purification and molecular properties of nitrite reductase from Anabaena sp. 7119. — Physiol. Plant. 52: 7–14.Google Scholar
  51. Miyata, M. and Mori, T. 1969. Studies on denitrification. X. The “denitrifying enzyme” as a nitrite reductase and the electron donating system for denitrification. — J. Biochem. (Tokyo) 66: 463–471.Google Scholar
  52. Nelson, L. M. and Knowles, R. 1978. Effect of oxygen and nitrate on nitrogen fixation and denitrification by Azospirillum brasilense grown in continuous culture. — Can. J. Microbiol. 24: 1395–1403.Google Scholar
  53. Newton, N. 1969. The two-haem nitrite reductase of Micrococcus denitrificans. — Biochim. Biophys. Acta 185: 316–331.Google Scholar
  54. Neyra, C. A., Dobereiner, J., Lalande, R. and Knowles, R. 1977. Denitrification by N2-fixing Spirillum lipoferum. — Can. J. Microbiol. 23: 300–305.Google Scholar
  55. Oltmann, L. F., Reijnders, W. N. M. and Stouthamer, A. H. 1976. Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis. — Arch. Microbiol. 111: 25–35.Google Scholar
  56. Palleroni, N. J., Kunisawa, R., Contopoulou, R. and Doudoroff, M. 1973. Nucleic acid homologies in the genus Pseudomonas. — Int. J. Syst. Bacteriol. 23: 333–339.Google Scholar
  57. Payne, W. J. 1973. Reduction of nitrogenous oxides by microorganisms. — Bacteriol. Rev. 37: 409–452.Google Scholar
  58. Payne, W. J., Riley, P. S. and Cox, C. D.Jr. 1971. Separate nitrite, nitric oxide, and nitrous oxide reducing fractions from Pseudomonas perfectomarinus. — J. Bacteriol. 106: 356–361.Google Scholar
  59. Pichinoty, F., de Barjac, H., Mandel, M., Greenway, B. and Garcia, J.-L. 1976a. Une nouvelle bactérie sporulée, dénitrifiante, mésophile: Bacillus azotoformans n. sp. — Ann. Microbiol. (Inst. Pasteur) 127B: 351–361.Google Scholar
  60. Pichinoty, F., Bigliardi-Rouvier, J., Mandel, M., Greenway, B., Metenier, G. and Garcia, J.-L. 1976b. The isolation and properties of a denitrifying bacterium of the genus Flavobacterium. — Antonie van Leeuwenhoek 42: 349–354.Google Scholar
  61. Pichinoty, F., Garcia, J.-L., Job, C. et Durand, M. 1978. La dénitrification chez Bacillus licheniformis. — Can. J. Microbiol. 24: 45–49.Google Scholar
  62. Pichinoty, F., Mandel, M. and Garcia, J.-L. 1977. Étude de six souches de Agrobacterium tumefaciens et A. radiobacter. — Ann. Microbiol. (Inst. Pasteur) 128A: 303–310.Google Scholar
  63. Renner, E. D. and Becker, G. E. 1970. Production of nitric oxide and nitrous oxide during denitrification by Corynebacterium nephridii. — J. Bacteriol. 101: 821–826.Google Scholar
  64. Ripley, E. M. and Nicol, D. L. 1981. Sulfur isotopic studies of Archean slate and graywacke from northern Minnesota: evidence for the existence of sulfate reducing bacteria. — Geochim. Cosmochim. Acta 45: 839–846.Google Scholar
  65. Rosso, J.-P., Forget, P. et Pichinoty, F. 1973. Les nitrate-reductases bactériennes. Solubilisation, purification et propriétés de l'enzyme A de Micrococcus halodénitrificans. — Biochim. Biophys. Acta 321: 443–455.Google Scholar
  66. Sadana, J. C., Khan, B. M. and Nicholas, D. J. D. 1981. Nitrite reductase from Achromobacter fischeri. — FEMS Microbiol. Lett. 12: 415–417.Google Scholar
  67. Satoh, T., Hoshino, Y. and Kitamira, H. 1976. Rhodopseudomonas sphaeroides forma sp. denitrificans, a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides. — Arch. Microbiol. 108: 265–269.Google Scholar
  68. Sawada, E., Satoh, T. and Kitamura, H. 1978. Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium. — Plant Cell. Physiol. 19: 1339–1351.Google Scholar
  69. Sawhney, V. and Nicholas, D. J. D. 1978. Sulphide-linked nitrite reductase from Thiobacillus denitrificans with cytochrome oxidase activity: purification and properties. — J. Gen. Microbiol. 106: 119–128.Google Scholar
  70. Schidlowski, M. 1979. Antiquity and evolutionary status of bacterial sulfate reduction: sulfur isotope evidence. — Origins Life 9: 299–311.Google Scholar
  71. Schwartz, R. M. and Dayhoff, M. O. 1978. Origens of prokaryotes, eukaryotes, mitochondria, and chloroplasts. — Science 199: 395–403.Google Scholar
  72. Seki-Chiba, S. and Ishimoto, M. 1977. Studies on nitrate reductase of Clostridium perfringens. I. Purification, some properties, and effect of tungstate on its formation. — J. Biochem. (Tokyo) 82: 1663–1671.Google Scholar
  73. Smibert, R. M. and Krieg, N. R. 1981. Nitrate reduction and denitrification. p. 419. In P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (eds), Manual of methods for general bacteriology. — Am. Soc. Microbiol. Washington, D. C.Google Scholar
  74. Smith, M. S. and Zimmerman, K. 1981. Nitrous oxide production by non-denitrifying soil nitrate reducers. — Soil Sci. Soc. Am. J. 45: 865–871.Google Scholar
  75. Snell, J. J. S. and Lapage, S. P. 1976. Transfer of some saccharolytic Moraxella species to Kingella, Hendriksen and Bøvre 1976, with descriptions of Kingella indologenes sp. nov. and Kingella denitrificans sp. nov. — Int. J. Syst. Bacteriol. 26: 451–458.Google Scholar
  76. Sperl, G. T. and Hoare, D. S. 1971. Denitrification with methanol: a selective enrichment for Hyphomicrobium species. — J. Bacteriol. 108: 733–736.Google Scholar
  77. Stanier, R. Y. 1947. Studies on non-fruiting myxobacteria. — J. Bacteriol. 53: 297–315.Google Scholar
  78. Stouthamer, A. H. 1980. Bioenergetic studies on Paracoccus denitrificans. — Trends Biochem. Sci. 5: 164–166.Google Scholar
  79. Strother, P. K. and Barghoorn, E. S. 1980. Microspheres from the Swartkoppie Formation: a review. p. 1–18. In H. O. Halvorson and K. E.Van Holde (eds). The origins of life and evolution. MBL Lectures in Biology, Vol. 1. — Alan R. Liss, Inc. New York.Google Scholar
  80. Terai, H. and Mori, T. 1975. Studies on phosphorylation coupled with denitrification and aerobic respiration in Pseudonomas denitrificans. — Bot. Mag. (Tokyo) 88: 231–244.Google Scholar
  81. Timmer-Ten Hoor, A. 1975. A new type of thiosulphate-oxidizing, nitrate-reducing microorganism: Thiomicrospira denitrificans sp. nov. — Neth. J. Sea Res. 9: 344–350.Google Scholar
  82. Timmer-Ten Hoor, A. 1981. Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans. — Antonie van Leeuwenhoek. 47: 231–243.Google Scholar
  83. Vangai, S. and Klein, D. A. 1974. Nitrite-dependent dissimilatory microorganisms isolated from Oregon soils. — Soil Biol. Biochem. 6: 335–339.Google Scholar
  84. Van Gent-Ruijters, M. L. W., De Vries, W. and Stouthamer, A. H. 1975. Influence of nitrate on fermentation pattern, molar growth yields and synthesis of cytochrome b in Propionibacterium pentosaceum. — J. Gen. Microbiol. 88: 36–48.Google Scholar
  85. Van't Riet, J., Wientjes, F. B., Van Doorn, J. and Planta, R. J. 1979. Purification and characterization of the respiratory nitrate reductase of Bacillus licheniformis. — Biochim. Biophys. Acta 576: 347–360.Google Scholar
  86. Vega, J. M., Guerrero, M. G., Leadbetter, E. and Losada, M. 1973. Reduced nicotinamide-adenine dinucleotide-nitrite reductase from Azotobacter chroococcum. — Biochem. J. 133: 701–708.Google Scholar
  87. Werber, M. M. and Mevarech, M. 1978. Induction of a dissimilatory reduction pathway of nitrate in Halobacterium of the Dead Sea. — Arch. Biochem. Biophys. 186: 60–65.Google Scholar
  88. Wesch, R. and Klemme, J.-H. 1980. Catalytic and molecular differences between assimilatory nitrate reductases isolated from two strains of Rhodopseudomonas capsulata. — FEMS Microbiol. Lett. 8: 37–41.Google Scholar
  89. Williams, R. J. and Evans, W. C. 1975. The metabolism of benzoate by Moraxella species through anaerobic nitrate respiration. — Biochem. J. 148: 1–10.Google Scholar
  90. Woese, C. R. 1980. An alternative to the Oparin view of the primeval sequence. p. 65–76. In H. O. Halvorson and K. E.Van Holde (eds), The origins of life and evolution. MBL Lectures in Biology, Vol. 1. — Alan R. Liss, Inc. New York.Google Scholar
  91. Yamanaka, T. and Okunuki, K. 1963. Crystalline Pseudomonas cytochrome oxidase. I. Enzymatic properties with special reference to the biological specificity. — Biochim. Biophys. Acta 67: 379–393.Google Scholar
  92. Zablotowicz, R. M. and Focht, D. D. 1979. Denitrification and anaerobic, nitrate-dependent acetylene reduction in cowpea Rhizobium. — J. Gen. Microbiol. 111: 445–448.Google Scholar
  93. Zohner, A. and Broda, E. 1979. Model experiments on nitrate and nitrate in simulated primeval conditions. — Origins Life 9: 291–298.Google Scholar

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© H. Veenman & Zonen 1982

Authors and Affiliations

  • Michael R. Betlach
    • 1
  1. 1.NASA Ames Research CenterMoffett FieldUSA

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