Applied Biochemistry and Biotechnology

, Volume 167, Issue 5, pp 1076–1091 | Cite as

Axenic Cultures of Nitrosomonas europaea and Nitrobacter winogradskyi in Autotrophic Conditions: a New Protocol for Kinetic Studies

  • B. Farges
  • L. Poughon
  • D. Roriz
  • C. CreulyEmail author
  • C.-G. Dussap
  • C. Lasseur


As a part of a natural biological N-cycle, nitrification is one of the steps included in the conception of artificial ecosystems designed for extraterrestrial life support systems (LSS) such as Micro-Ecological Life Support System Alternative (MELiSSA) project, which is the LSS project of the European Space Agency. Nitrification in aerobic environments is carried out by two groups of bacteria in a two-step process. The ammonia-oxidizing bacteria (Nitrosomonas europaea) realize the oxidation of ammonia to nitrite, and the nitrite-oxidizing bacteria (Nitrobacter winogradskyi), the oxidation of nitrite to nitrate. In both cases, the bacteria achieve these oxidations to obtain an energy and reductant source for their growth and maintenance. Furthermore, both groups also use CO2 predominantly as their carbon source. They are typically found together in ecosystems, and consequently, nitrite accumulation is rare. Due to the necessity of modeling accurately conversion yields and transformation rates to achieve a complete modeling of MELiSSA, the present study focuses on the experimental determination of nitrogen to biomass conversion yields. Kinetic and mass balance studies for axenic cultures of Nitrosomonas europaea and Nitrobacter winogradskyi in autotrophic conditions are performed. The follow-up of these cultures is done using flow cytometry for assessing biomass concentrations and ionic chromatography for ammonium, nitrite, and nitrate concentrations. A linear correlation is observed between cell count and optical density (OD) measurement (within a 10 % accuracy) validating OD measurements for an on-line estimation of biomass quantity even at very low biomass concentrations. The conversion between cell count and biomass concentration has been determined: 7.1 × 1012 cells g dry matter (DM)−1 for Nitrobacter and 6.3 × 1012 cells g DM−1 for Nitrosomonas. Nitrogen substrates and products are assessed redundantly showing excellent agreement for mass balance purposes and conversion yields determination. Although the dominant phenomena are the oxidation of NH 4 + into nitrite (0.95 mol mol N−1 for Nitrosomonas europaea within an accuracy of 3 %) and nitrite into nitrate (0.975 mol mol N−1 for Nitrobacter winogradskyi within an accuracy of 2 %), the Nitrosomonas europaea conversion yield is estimated to be 0.42 g DM mol N−1, and Nitrobacter winogradskyi conversion yield is estimated to be 0.27 g DM mol N−1. The growth rates of both strains appear to be dominated by the oxygen transfer into the experimental setups.


Autotrophy Nitrosomonas europaea Nitrobacter winogradskyi Ionic chromatography Flow cytometry 



The authors thank the European Space Agency for the financial support of this work


  1. 1.
    Godia, F., Albiol, J., Montesinos, J. L., Pérez, J., Creus, N., Cabello, F., Mengual, X., Montras, A., & Lasseur, Ch. (2002). Journal of Biotechnology, 99, 319–330.CrossRefGoogle Scholar
  2. 2.
    Hendrickx, L., De Wever, H., Hermans, V., Mastroleo, F., Morin, N., Wilmotte, A., Janssen, P., & Mergeay, M. (2006). Research in Microbiology, 157(1), 77–86.CrossRefGoogle Scholar
  3. 3.
    Pérez, J., Poughon, L., Dussap, C. G., Montesinos, J. L., & Godia, F. (2005). Process Biochemistry, 40, 2359–2369.CrossRefGoogle Scholar
  4. 4.
    Wittebolle, L., Verstraete, W., & Boon, N. (2009). Water Research, 43, 4149–4158.CrossRefGoogle Scholar
  5. 5.
    Delatolla, R., Tufenkji, N., Comeau, Y., Lamarre, D., Gadbois, A., & Berk, D. (2009). Water Research, 43, 1775–1787.CrossRefGoogle Scholar
  6. 6.
    Dytczak, M. A., Londry, K. L., & Oleszkiewicz, J. A. (2008). Water Research, 42, 2320–2328.CrossRefGoogle Scholar
  7. 7.
    Haseborg, E., Zamora, T. M., Fröhlich, J., & Frimmel, F. H. (2010). Bioresource Technology, 101, 1701–1706.CrossRefGoogle Scholar
  8. 8.
    Vadivelu, V. M., Keller, J., & Yuan, Z. (2007). Water Research, 41, 826–834.CrossRefGoogle Scholar
  9. 9.
    Bock, E. (1976). Archives of Microbiology, 108, 305–312.CrossRefGoogle Scholar
  10. 10.
    Sarioglu, M., Insel, G., Artan, N., & Orhon, D. (2011). Journal of Chemical Technology and Biotechnology, 86, 798–811.CrossRefGoogle Scholar
  11. 11.
    Chen, R. D., & LaPara, T. M. (2008). Process Biochemistry, 43, 33–41.CrossRefGoogle Scholar
  12. 12.
    Prosser, J. I., & Embley, T. M. (2002). Antonie Van Leeuwenhoek, 81, 165–179.CrossRefGoogle Scholar
  13. 13.
    Laanbroek, H. J., Bär-Gilissen, M. J., & Hoogveld, H. L. (2002). Applied and Environmental Microbiology, 68, 1454–1457.CrossRefGoogle Scholar
  14. 14.
    Chapman, B. D., Schleicher, M., Beuger, A., Gostomski, P., & Thiele, J. H. (2006). Journal of Microbiological Methods, 65, 96–106.CrossRefGoogle Scholar
  15. 15.
    Güven, D., & Schmidt, I. (2009). Process Biochemistry, 44, 516–520.CrossRefGoogle Scholar
  16. 16.
    Anthonisen, D. J., Loehr, R. C., Prakasam, T. B. S., & Srinath, E. G. (1976). Journal of the Water Pollution Control Federation, 48, 835–852.Google Scholar
  17. 17.
    Groeneweg, J., Sellner, B., & Tappe, W. (1994). Water Research, 28, 2561–2566.CrossRefGoogle Scholar
  18. 18.
    Arp, D. J., & Stein, L. Y. (2003). Critical Reviews in Biochemistry and Molecular Biology, 38, 471–495.CrossRefGoogle Scholar
  19. 19.
    Princic, A., Mahne, I. I., Megusar, F., Paul, E. A., & Tiedje, J. M. (1998). Applied and Environmental Microbiology, 64, 3584–3590.Google Scholar
  20. 20.
    Grady, C. P. L., & Lim, H. C. (1980). Biological wastewater treatment, theory and applications (pp. 291–299). New York: Marcel Dekker.Google Scholar
  21. 21.
    Junier, P., Molina, V., Dorador, C., Hadas, O., Kim, O. S., Junier, T., Witzel, K. P., & Imhoff, J. F. (2010). Applied Microbiology and Biotechnology, 85(3), 425–440.CrossRefGoogle Scholar
  22. 22.
    Haug, R. T., & Mc Carty, P. L. (1972). Journal of the Water Pollution Control Federation, 44, 2086.Google Scholar
  23. 23.
    Montras, A., Pycke, B., Boon, N., Godia, F., Mergeay, M., Hendrickx, L., & Perez, J. (2008). Water Research, 42, 1700–1714.CrossRefGoogle Scholar
  24. 24.
    Stein, L., & Arp, D. J. (1998). Applied and Environmental Microbiology, 64, 4098–4102.Google Scholar
  25. 25.
    Bock, E., Koops, H. P., & Harms, H. (1989). In H. G. Schlegel & B. Bowien (Eds.), Autotrophic bacteria (pp. 81–96). Berlin: Springer-Verlag.Google Scholar
  26. 26.
    Patton, C. J., & Crouch, S. R. (1977). Analytical Chemistry, 49(3), 464–469.CrossRefGoogle Scholar
  27. 27.
    Loveless, J. E., & Painter, H. A. (1968). Journal of General Microbiology, 52, 1–14.CrossRefGoogle Scholar
  28. 28.
    Skinner, F. A., & Walker, N. (1961). Archiv für Mikrobiologie, 38, 339–349.CrossRefGoogle Scholar
  29. 29.
    Drozd, J. W. (1980). In C. J. Knowles (Ed.), Diversity of bacterial respiratory systems (Vol. 2, pp. 87–111). Boca Raton: CRC.Google Scholar
  30. 30.
    Belser, L. W., & Schmidt, E. L. (1980). FEMS Microbiology Letters, 7, 213–216.CrossRefGoogle Scholar
  31. 31.
    Helder, W., & De Vries, R. T. P. (1983). Netherlands Journal of Sea Research, 17, 1–18.CrossRefGoogle Scholar
  32. 32.
    Keen, G. A., & Prosser, J. I. (1987). Archives of Microbiology, 147, 73–79.CrossRefGoogle Scholar
  33. 33.
    Glover, H. E. (1985). Archives of Microbiology, 142, 45–50.CrossRefGoogle Scholar
  34. 34.
    Hunik, J. H., Bos, C. G., den Hoogen, M. P., De Gooijer, C. D., & Tramper, J. (1994). Biotechnology and Bioengineering, 43, 1153–1163.CrossRefGoogle Scholar
  35. 35.
    Brion, N., & Billen, G. (1998). Revue des Sciences de l'eau, 11, 283–302.Google Scholar
  36. 36.
    Kantartartzi, S. G., Vaiopoulos, E., Kapagiannidis, A., & Aivasidis, A. (2006). Global NEST Journal, 8, 43–51.Google Scholar
  37. 37.
    Fang, F., Bing-Jie, N., Xiao-Yan, L., Guo-Ping, S., & Han-Qing, Y. (2009). Applied Microbiology and Biotechnology, 83, 1159–1169.CrossRefGoogle Scholar
  38. 38.
    Park, H. D., & Noguera, D. R. (2007). Journal of Applied Microbiology, 102, 1401–1417.CrossRefGoogle Scholar
  39. 39.
    Gould, G. W., & Lees, H. (1960). Canadian Journal of Microbiology, 6, 299–307.CrossRefGoogle Scholar
  40. 40.
    Gay, G., & Corman, A. (1984). Microbial Ecology, 10, 99–105.CrossRefGoogle Scholar
  41. 41.
    Cox, D. J., Bazin, M. J., & Gull, K. (1980). Soil Biology and Biochemistry, 12, 241–246.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • B. Farges
    • 1
  • L. Poughon
    • 1
  • D. Roriz
    • 1
  • C. Creuly
    • 1
    Email author
  • C.-G. Dussap
    • 1
  • C. Lasseur
    • 2
  1. 1.Laboratoire de Génie Chimique et Biochimique, Polytech Clermont-FerrandClermont UniversitéAubière cedexFrance
  2. 2.ESTECEuropean Space AgencyNoordwijkThe Netherlands

Personalised recommendations