Abstract
The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional ‘cultivation based techniques’ alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers.
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References
Broda E (1977) Two kinds of lithotrophs missing in nature. Z. Allg. Mikrobiol. 17: 491–493.
Bryant MP, Wolin EA, Wolin MJ & Wolfe RS (1967) Methanobacillus omelanskii, a symbiotic association of two species of bacteria. Arch. Microbiol. 59: 20–31.
Daims H, Bruhl A, Amann M, Schleifer KH & Wagner M (1999) The domain specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probeset. Syst. Appl. Microbiol. 22: 434–444.
Egli K, Franger U, Alvarez PJJ, Siegrist H, Van der Meer JR & Zehnder AJB (2001) Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Arch. Microbiol. 175: 198–207.
Helmer C & Kunst S (1998) Simultaneous nitrification/denitrification in an aerobic biofilm system. Water Sci. Technol. 37: 183–187.
Jetten MSM, Strous M, Van de Pas-Schoonen KT, Schalk J, Van Dongen LGJM, Van de Graaf AA, Logemann S, Muyzer G, Van Loosdrecht MCM & Kuenen JG (1999) The anaerobic oxidation of ammonium. FEMS Microbiol. Rev. 22: 421–437.
Juretschko S, Timmermann G, Schmid M, Schleifer KH, Pommereningroser A, Koops HP & Wagner M (1998) Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge - nitrosococcus mobilis and nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol. 64: 3042–3051.
Lindsay MR, Webb RI, Strous M, Jetten MS, Butler MK, Forde RJ & Fuerst JA (2001) Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell. Arch. Microbiol. 175(6): 413–429.
McKinley JP, Stevens PO, Long PE, Balkwill DL, Griffin WT & Kieft T (1998) Observations pertaining to the origin and ecology of microorganisms recovered from the deep subsurface of Taylorsville Basin, Virginia. Geomicrobiol. J. 15(4): 353–386.
Mulder A, Van de Graaf AA, Robertson LA & Kuenen JG (1995) Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16: 177–183.
Murray JW, Jannash HW, Honjo S, Anderson RF, Reeburgh WS, Top Z, Friederich GE, Lodispoti LA & Izdar E (1989) Unexpected changes in the oxic/anoxic interface in the Black Sea. Nature 338: 411–413.
Orphan VJ, House CH, Hinrichs KU, McKeegan KD & DeLong EF (2001) Methane-consuming Archaea revealed by directly coupled isotopic and phylogenetic analysis. Science 293: 484–487.
Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276: 734–740.
Schalk J, De Vries S, Kuenen JG & Jetten MSM (2000) Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation. Biochemistry 39(18): 5405–5412.
Schmid M, Twachtmann U, Klein M, Strous M, Juretschko S, Jetten MSM, Metzger JW, Schleifer KH & Wagner M (2000) Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst. Appl. Microbiol. 23: 93–106.
Schmid M, Schmitz-Esser S, Jetten MSM & Wagner M (2001) 16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium oxidizing bacteria: implications for phylogeny and in situ detection. Env. Microbiol. 3(7): 450–459.
Schulz HN, Brinkhoff T, Ferdelman TG, Hernandez Marine M, Teske A & Jorgensen BB (1999) Dense populations of a giant sulfur bacterium in Namibian shelf sediments. Science 284: 493–495.
Siegrist H, Reithaar S, Koch G & Lais P (1998) Nitrogen loss in a nitrifying rotating contactor treating ammonium-rich wastewater without organic carbon. Water. Sci. Technol. 38: 241–248.
Strous M, Van Gerven E, Ping Z, Kuenen JG & Jetten MSM (1997) Ammonium removal from concentrated waste streams with the Anaerobic Ammonium Oxidation (Anammox) process in different reactor configurations. Water. Res. 31: 1955–1962.
Strous M, Heijnen JJ, Kuenen JG & Jetten MSM (1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50: 589–596.
Strous M, Fuerst JA, Kramer EHM, Logemann S, Muyzer G, Van de Pas-Schoonen KT, Webb R, Kuenen JG & Jetten MSM (1999a) Missing lithotroph identified as new planctomycete. Nature 400: 446–449.
Strous M, Kuenen JG & Jetten MSM (1999b) The key physiological parameters of the anaerobic ammonium oxidation process. Appl. Environ. Microbiol. 65: 3248–3250.
Strous, M. Microbiology of anaerobic ammonium oxidation. PhD Thesis. TU Delft. 2000.
Thamdrup B & Dalsgaard T (2002) Production of N 2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl. Environ. Microbiol. 60: 1312–1318.
Van de Graaf AA, De Bruijn P, Robertson LA, Jetten MSM & Kuenen JG (1996) Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology (UK) 142: 2187–2196.
Van de Graaf AA, Mulder A, De Bruijn P, Jetten MSM, Robertson LA & Kuenen JG (1995) Anaerobic oxidation of ammonium is a biologically mediated process. Appl. Environ. Microbiol. 61: 1246–1251.
Van Loosdrecht MCM, Smolders GJ, Kuba T & Heijnen JJ (1997) Metabolism of micro-organisms responsible for enhanced biological phosphorus removal from wastewater - Use of dynamic enrichment cultures. Anton. Leeuwenhoek Int. J. Gen. M. 71: 109–116.
Whitman WB, Coleman DC & Wiebe WJ (1998) Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. USA 95: 6578–6583.
Winogradsky S (1949) Microbiology du sol - problèmes et méthodes - cinquantes ans de recherches. Paris, Masson et cie éditeurs.
Woese CR (1987) Bacterial evolution. Microbiol. Rev. 51: 221–271.
Zuckerlandl E & Pauling L (1965) J. Theor. Biol. 8: 357–366.
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Strous, M., Kuenen, J.G., Fuerst, J.A. et al. The anammox case – A new experimental manifesto for microbiological eco-physiology. Antonie Van Leeuwenhoek 81, 693–702 (2002). https://doi.org/10.1023/A:1020590413079
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DOI: https://doi.org/10.1023/A:1020590413079