FEMS Microbiology Ecology 39 (2002) 175^181

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Aerobic and anaerobic ammonia oxidizing bacteria ^ competitors or natural partners?

Ingo Schmidt *, Olav Sliekers b , Markus Schmid b , Irina Cirpus b , Marc Strous a , Eberhard Bock c , J. Gijs Kuenen b , Mike S.M. Jetten a
a;

a University of Nijmegen, Department of Microbiology, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Delft University of Technology, Kluyver Laboratory for Biotechnology, Department of Microbiology and Enzymology, Julianalaan 67, 2628 BC Delft, The Netherlands c University of Hamburg, Institute for Botany, Department of Microbiology, OhnhorststraMe 18, 22609 Hamburg, Germany

Received 20 September 2001; received in revised form 16 November 2001; accepted 20 November 2001 First published online 21 December 2001

Abstract
The biological nitrogen cycle is a complex interplay between many microorganisms catalyzing different reactions. For a long time, ammonia and nitrite oxidation by chemolithoautotrophic nitrifiers were thought to be restricted to oxic environments and the metabolic flexibility of these organisms seemed to be limited. The discovery of a novel pathway for anaerobic ammonia oxidation by Planctomyces (anammox) and the finding of an anoxic metabolism by classical Nitrosomonas-like organisms showed that this is no longer valid. The aim of this review is to summarize these novel findings in nitrogen conversion and to discuss the ecological importance of these processes. 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Nitrication; Anammox ; Candidatus Brocadia anammoxidans ; NOx cycle; Nitrosomonas ; Aerobic ammonia oxidation; Anaerobic ammonia oxidation

1. Introduction Nitrication is an important part of the biological nitrogen cycle. Microorganisms involved in nitrication are characterized as lithotrophic ammonia and nitrite oxidizing bacteria and heterotrophic nitriers (not discussed in this review). Lithotrophic nitriers are all placed in the family Nitrobacteraceae [1], although they are not necessarily related phylogenetically. Chemolithoautotrophic nitrifying bacteria have been found in many ecosystems such as fresh water, salt water, sewage systems, soils, and on/in rocks as well as in masonry [2,3]. Growth under suboptimal conditions might be possible by ureolytic activity, aggregate formation [4], or in biolms on the surfaces of substrata [5]. Nitriers can be found in extreme habitats at high temperatures [6] and in Antarctic soils [7,8]. Although the pH optimum for cell growth is pH 7.6^7.8, they were frequently detected in environments with pH

values of about 4 such as acid tea and forest soils [9,10] and pH values of about 10 such as soda lakes [11,12]. It is interesting to note that aerobic nitriers were also found in anoxic environments [13,14]. This is in good agreement with recent studies that show that these microorganisms have a more versatile metabolism than previously assumed. Ammonia oxidizers can denitrify with ammonia as electron donor under oxygen-limited conditions [15,16] or with hydrogen or organic compounds under anoxic conditions [17]. Finally they can use N2 O4 as oxidant for ammonia oxidation under both oxic and anoxic conditions [18]. Furthermore, a new group of anaerobic nitrite-dependent ammonia oxidizers (anammox) were discovered [19,20]. This review will discuss the recent ndings and their ecological importance for the understanding of the biological nitrogen cycle.

2. Anaerobic ammonium oxidation (anammox)

* Corresponding author. Tel. : +31 (24) 3652568; Fax: +31 (24) 3652830. E-mail address : i.schmidt@sci.kun.nl (I. Schmidt).

2.1. Molecular identity Although Broda [21] predicted the existence of chemo-

0168-6496 / 01 / $22.00 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 2 0 8 - 2

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lithoautotrophic bacteria capable of anaerobic ammonium oxidation and Abeliovich [14] reported high cell concentrations of nitriers under anoxic conditions, the rst experimental conrmation of anaerobic ammonia oxidation (anammox) was obtained in the early 1990s [19]. During experiments on a denitrifying pilot plant it was noted that ammonia and nitrate disappeared from the reactor euent with a concomitant increase of dinitrogen gas production. The microbial nature of the process was veried, and nitrite was shown to be the preferred electron acceptor [22]. Hydroxylamine and hydrazine were identied as important intermediates. Since the growth rate of the anammox biomass appeared to be very low (doubling time about 11 days), reactor systems with very ecient biomass retention were necessary for the enrichment. A sequencing batch reactor system was chosen for the ecophysiological study of the anammox community [23]. The biomass in the community was dominated for more than 70% by a morphologically conspicuous bacterium. Attempts to isolate the microorganisms with classical methods failed. Therefore, the bacterium was physically puried from enrichment cultures by density gradient centrifugation [24]. DNA extracted from the puried cells was used as a template for PCR amplication with a universal 16S rDNA primer set. The dominant 16S rDNA sequence obtained was planctomycete-like, and branching very deep within the planctomycete lineage of descent (Fig. 1). The anaerobic ammonium oxidizing planctomycete-like bacterium was named Candidatus Brocadia anammoxidans. The 16S rDNA sequence information was used to design specic oligonucleotide probes for application in uorescence in situ hybridization (FISH) and to survey the presence of B. anammoxidans and related anammox bacteria in several

wastewater treatment systems [25]. Indeed, B. anammoxidans and the closely related Candidatus Kuenenia stuttgartiensis could be detected in many of these systems throughout the world and seem to be dominating in these microbial biolm communities [25]. 2.2. Molecular diversity The order Planctomycetales, rst described in 1986 by Schlessner and Stackebrandt [26], so far includes only four genera (Planctomyces, Pirellula, Gemmata, and Isosphaera) with seven validly described species [27]. Various environmentally derived 16S rDNA sequences [20,28] strongly indicate further planctomycete lineages [29], including the anammox bacteria (Fig. 1). In fact, the newly found bacterium K. stuttgartiensis forms a distinct branch within anammox bacteria and the sequence similarity of less than 90% to B. anammoxidans is indicative of a genus level diversity of these bacteria [25]. The application of FISH probes showed the dominance of these bacteria in ecosystems with high nitrogen losses. Molecular techniques are important tools to monitor the presence and activity of microorganisms in ecosystems. For example the growth rate of many bacteria can be deduced from their ribosome content [30]. This method is, however, not applicable for slow-growing anammox- and Nitrosomonas-like bacteria [31] since inactive cells of both groups tend to keep their ribosome content at a high level. In such cases, the cellular concentrations of precursor rRNA might be a good indicator of physiological activity [32]. Therefore, the intergenic spacer regions (ISR) between the 16S rRNA and 23S rRNA, as part of the precursor rRNA, of B. anammoxidans and K. stuttgartiensis were sequenced. Subse-

Fig. 1. 16S rDNA-based phylogenetic dendrogram reecting the relationships of Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans to organisms aliated to the order Planctomycetales. The tree is based on results of maximum likelihood analyses from dierent data sets. The black bars indicate phylogenetic groups. Environmentally derived sequences mainly originating from the Antarctic were pooled in the Antarctic clone cluster. GenBank accession numbers are given in parentheses. The bar represents 10% estimated sequence divergence.

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quently, ISR-targeted oligonucleotide probes were constructed and applied by FISH. Inhibition experiments with B. anammoxidans revealed a good correlation between the metabolic activity and the ISR concentrations, demonstrating the ISR targeting FISH to be a powerful method for the detection of activity changes in slow-growing bacteria [31]. 2.3. Ecophysiology The ultrastructure of B. anammoxidans has many features in common with previously described planctomycetes. These microorganisms have a proteinaceous cell wall lacking peptidoglycan and are thus insensitive to ampicillin. The chromosome is separated from the surrounding cytoplasm by a single or double membrane. In B. anammoxidans an additional compartment bounded by a single membrane [33], free from ribosomes and chromosome, was observed. This peculiar organelle made up more than 30% of the cell volume and it may play an important role in the catabolism. Using the immunogold labeling technique with antibodies against the key enzyme hydroxylamine (hydrazine) oxidoreductase [34], the enzyme was localized in this middle compartment, which was named anammoxosome [33]. Interestingly, B. anammoxidans [33] as well as aerobic ammonia oxidizers such as Nitrosomonas [1] develop internal membrane systems. Whether such a membrane system is bioenergetically necessary for ammonia oxidation is still the subject of investigation since both key enzymes of Nitrosomonas are obviously not localized in the intracytoplasmic membrane (ICM) system. According to the peptide structure the AMO was described as a membrane-bound enzyme [35], and recent studies [36] indicated a localization in the cytoplasmic membrane. The HAO is localized in the periplasm [37]. To unravel the metabolic pathway for anaerobic ammonium oxidation in B. anammoxidans, series of 15 N-labeling experiments were conducted. It could be shown that ammonium and nitrite are combined to yield dinitrogen gas [38] and radioactive bicarbonate is incorporated in the biomass. With an excess of hydroxylamine, a transient accumulation of hydrazine was observed, indicating that hydrazine is an intermediate of the anammox process. According to the working hypothesis, the oxidation of hydrazine to dinitrogen gas is supposed to generate four electrons for the initial reduction of nitrite to hydroxylamine (Fig. 2). The overall nitrogen balance shows a ratio of about 1:1.32:0.26 for the conversion of ammonia, nitrite, and nitrate (Eq. 1). The function of the formation of nitrate is assumed to be the generation of reducing equivalents necessary for the reduction of CO2 .
NH 1:32 NO3 0:066 HCO3 0:13 H ! 0:26 NO3 2 3 3 4 1:02 N 2 0:066 CH 2 O0:5 N 0:15 2:03 H 2 O
Fig. 2. Proposed model for the anaerobic ammonia oxidation (anammox) of Brocadia-like microorganisms. HH: hydrazine hydrolase ; HZO : hydrazine oxidizing enzyme; NR : nitrite reducing enzyme.

A high anammox activity is detectable in a pH range between 6.4 and 8.3 and a temperature range between 20 and 43C [39]. Under optimal conditions, the specic activity is about 3.6 mmol (g protein)31 h31 , the biomass yield about 0.066 C-mol (mol ammonium)31 , and the specic growth rate about 0.0027 h31 . Recent studies showed that K. stuttgartiensis is in many ways similar to B. anammoxidans [40]. K. stuttgartiensis cells have the same overall cell structure and also produce hydrazine from exogenously supplied hydroxylamine. Energetically favorable mechanisms with Fe3 , Mn4 , or even sulfate as oxidant have not been reported yet [41]. To assess the occurrence of the anammox reaction in natural environments and man-made ecosystems, further data about the eect of several chemical and physical parameters are necessary. For example the anammox bacteria are very sensitive to oxygen and nitrite. Oxygen concentrations as low as 2 WM and nitrite concentrations between 5 and 10 mM inhibit the anammox activity completely, but reversibly [22].

3. Ecology of anammox In various ecosystems B. anammoxidans will be dependent on the activity of aerobic ammonia oxidizing bacteria under oxygen-limited conditions, e.g., at the oxic/anoxic interface. Anammox biomass has already been detected in wastewater treatment plants in The Netherlands, Germany, Switzerland, UK, Australia, and Japan [42]. Recently anammox cells were detected in a non-articial ecosystem, a fresh water swamp in Uganda [42]. Oxic/anoxic interfaces are abundant in nature, for example in biolms and ocs. In these oxygen-limited environments the ammonia oxidizers would oxidize ammonium to nitrite and keep the oxygen concentration low, while B. anammoxidans would convert the produced nitrite and the remaining ammonium to dinitrogen gas. Such conditions have been established in many dierent reactor systems [16,43^45]. FISH analysis and activity measurements showed that

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aerobic as well as anaerobic ammonia oxidizers were present and active in these oxygen-limited reactors, but aerobic nitrite oxidizers (Nitrobacter or Nitrospira) were not detected. Apparently, the aerobic nitrite oxidizers are unable to compete for oxygen with the aerobic ammonia oxidizers and for nitrite with the anaerobic ammonia oxidizers as has been documented before [46,47]. It seems likely that under these conditions anaerobic and aerobic ammonia oxidizers form a quite stable community. The cooperation of aerobic and anaerobic ammonium oxidizing bacteria is not only relevant for wastewater treatment [45,48], but might play an important role in natural environments at the oxic/anoxic interface. Further interactions under anoxic conditions between both groups of ammonia oxidizers seem to be likely since an anoxic, NO2 -dependent metabolism of Nitrosomonas-like microorganisms was recently discovered [18].

NH3 N2 O4 2 H 2 e3 ! NH2 OH H2 O 2 NO 2 NH2 OH H2 O ! HNO2 4 H 4 e3 NH3 N2 O4 ! HNO2 2 NO 2 H 2 e3 3 4

Hydroxylamine and nitric oxide are formed in this reaction. While nitric oxide is not further metabolized, hydroxylamine is oxidized to nitrite. The nitrite produced is partly used as electron acceptor leading to the formation of dinitrogen: HNO2 3 H 3 e3 ! 0:5 N2 2 H2 O 5

4. Aerobic and anaerobic NO2 -dependent ammonia oxidation by Nitrosomonas (NOx cycle) 4.1. Diversity Gram-negative ammonia oxidizers, e.g., members of the genera Nitrosomonas and Nitrosospira [1], are lithoautotrophic organisms using carbon dioxide as the main carbon source. Several species reveal extensive ICM systems. Recently, molecular tools to detect the presence of ammonia oxidizing bacteria in the environment have been supplemented by PCR primers for specic amplication of the ammonia monooxygenase structural gene amoA [3]. Environmental 16S rRNA and amoA libraries have extended the knowledge on the natural diversity of ammonia oxidizing bacteria [49]. Comparative 16S rRNA sequence analyses revealed that members of this physiological group are conned to two monophyletic lineages within the Proteobacteria. Nitrosococcus oceanus is aliated with the Q-subclass of the Proteobacteria, while members of the genera Nitrosomonas and Nitrosospira form a closely related group within the L-subclass of Proteobacteria [50]. Using these molecular tools nitriers can be detected even in anoxic habitats. 4.2. Anaerobic ammonia oxidation Recently published data gave rst evidence for anaerobic ammonia oxidation by Nitrosomonas [51]. These results indicate a complex role of nitrogen oxides (NO and NO2 ) in the metabolism of aerobic ammonia oxidizers. Nitrosomonas eutropha can oxidize ammonia in the absence of dissolved oxygen [51,52], replacing molecular oxygen by nitrogen dioxide or nitrogen tetroxide (dimeric form of NO2 ). The overall nitrogen balance shows a ratio of about 1:1:1:2 for the conversion of ammonia, nitrogen tetroxide, nitrite, and nitric oxide:

There are only a few dierences between the anaerobic, NO2 -dependent and the aerobic, O2 -dependent [53] ammonia oxidation by Nitrosomonas. Instead of O2 in the course of aerobic ammonia oxidation, N2 O4 is used as electron acceptor and NO, an additional product, is released in the anaerobic ammonia oxidation. NO2 is not available in natural environments under anoxic conditions. An anaerobic ammonia oxidation is therefore dependent on the transport of NO2 from oxic layers. Another important observation is that anaerobic ammonia oxidation with NO2 (N2 O4 ) as oxidant was not affected by acetylene [18]. N. eutropha cells treated with acetylene oxidized ammonia even under oxic conditions if NO2 was available. Ammonia oxidation was not detectable in the absence of NO2 . One of the most signicant ndings is that the 27-kDa polypeptide of the AMO was not labeled with [14 C]acetylene during anoxic NO2 -dependent ammonia oxidation. When oxygen was added, the labeling of this polypeptide with [14 C]acetylene started immediately. An inuence of the ammonia concentration on the labeling reaction was not observed. These studies clearly demonstrate the necessity to distinguish between NO2 -dependent and O2 -dependent ammonia oxidation. The new hypothetical model of ammonia oxidation [18] including the role of nitrogen oxides is shown in Fig. 3. Anaerobic ammonia oxidation is dependent on the pres-

Fig. 3. NOx cycle. Hypothetical model of the anaerobic NO2 -dependent ammonia oxidation by Nitrosomonas. N2 O4 is the oxidant for the ammonia oxidation.

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ence of the oxidizing agent N2 O4 . NO is produced in stoichiometric amounts (Fig. 3). Only when NO2 is available under anoxic conditions, ammonia is oxidized and hydroxylamine occurs as an intermediate while NO is formed as an end product. Hydroxylamine is further oxidized to nitrite [52]. Under anoxic conditions nitrite serves as a terminal electron acceptor. In the absence of ammonia Nitrosomonas is capable of using dierent substrates as electron donor. During hydroxylamine oxidation by ammonia oxidizers, small amounts of nitric and nitrous oxide are released [54]. Both gases are also produced in the course of aerobic denitrication by ammonia oxidizing bacteria [55,56]. Additionally, the formation of dinitrogen was observed [17,57]. Furthermore, Nitrosomonas is capable of anoxic denitrication with molecular hydrogen [17] or simple organic compounds [14] serving as electron donors. 4.3. Aerobic ammonia oxidation NOx also plays an important role in the aerobic metabolism of nitrifying microorganisms. Nitrosomonas-like organisms were distinctly inhibited when gaseous nitric oxide was removed from laboratory-scale cultures by means of intensive aeration. Nitrication in these cultures only started again when nitric oxide was added to the gas inlet of the culture vessels [18,58]. The lag phase during the recovery of ammonia oxidation in starved cells could be signicantly reduced when NOx was added. Evidence is given that the cells generate NO for the NOx cycle via denitrication when external NOx is not available [59]. Nitrogenous oxides have a signicant promoting eect on pure cultures of N. eutropha [59,60]. Their addition resulted in a pronounced increase in nitrication rate, specic activity of ammonia oxidation, growth rate, maximum cell density, and aerobic denitrication capacity. Maximum cell numbers amounted to 2U1010 Nitrosomonas cells ml31 . Furthermore, about 50% of the nitrite produced was aerobically denitried to dinitrogen when nitrogen oxides were present. In the presence of O2 , the produced NO can be (re)oxidized to NO2 [18]. Therefore, only small amounts of NO are detectable in the gas phase of Nitrosomonas cell suspensions. According to the model (Fig. 4, Eq. 6), N2 O4 is the oxidizing agent under oxic conditions. Hydroxylamine and NO are produced as intermediates. While hydroxylamine is further oxidized to nitrite, NO is (re)oxidized to NO2 (N2 O4 ) (Eq. 7): NH3 N2 O4 2 H 2 e3 ! NH2 OH 2 NO H2 O 6 2NO O2 ! 2 NO2 N2 O4 NH3 O2 2 H 2 e3 ! NH2 OH H2 O 7 8

Fig. 4. NOx cycle. Hypothetical model of the ammonia oxidation by Nitrosomonas. According to this model, N2 O4 is the oxidant for the ammonia oxidation. Under oxic conditions oxygen is used to re-oxidize NO to NO2 (N2 O4 ). Hydroxylamine is oxidized to nitrite.

described earlier as the reaction of aerobic ammonia oxidation [53], but is in complete agreement with the new hypothetical model. The total consumption rates (ammonia, oxygen) and production rates (hydroxylamine as intermediate) are the same, but the mechanism of the reaction is quite dierent. Since detectable NOx concentrations were small, nitrogen oxides seem to cycle in the cell (possibly enzyme-bound). Therefore, the total amount of NOx per cells is expected to be low. This hypothetical model (Fig. 4) is in good accordance with the described mechanisms of the aerobic ammonia oxidation. According to the new model, O2 is used to oxidize NO. The product NO2 is then consumed during ammonia oxidation. The oxygen of hydroxylamine still originates from molecular oxygen, but is incorporated via NO2 [18]. In control experiments dierent species of ammonia oxidizers were tested (e.g., Nitrosomonas europaea, Nitrosolobus multiformis) [61]. All species were able to oxidize ammonia under anoxic conditions with NO2 as oxidant (Fig. 3) and the aerobic ammonia oxidation activity was increased in the presence of NO or NO2 (Fig. 4). 4.4. Ecological evidence of NOx The ecological evidence of nitrogen oxides (NOx ) for nitrication is still object of speculations and there is no simple, uniform picture of the function of NOx in the ammonia oxidation. Further investigations are necessary to reveal the role of nitrogen oxides. First, NO2 has to be conrmed as the master regulating signal for the ammonia oxidation [59]. The recovery of ammonia oxidation activity by denitrifying Nitrosomonas cells (hydrogen as electron donor) is regulated via the availability of NO2 . Second, in contrast to homoserine lactones, which function as signal molecules between many bacteria [62], nitrogen oxides seem to function as very specic signal molecules between ammonia oxidizers [59].

The sum of Eqs. 6 and 7, given in Eq. 8, was already

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I. Schmidt et al. / FEMS Microbiology Ecology 39 (2001) 175^181 oxidizing bacteria from Mongolian soda lakes. Arch. Microbiol. 176, 170^177. Weber, S., Stubner, S. and Conrad, R. (2001) Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl. Environ. Microbiol. 67, 1318^1327. Abeliovich, A. and Vonhak, A. (1992) Anaerobic metabolism of Nitrosomonas europaea. Arch. Microbiol. 158, 267^270. Goreau, T.J., Kaplan, W.A., Wofsy, S.C., McElroy, M.B., Valois, F.W. and Watson, S.W. (1980) Production of NO3 and N2 O by 2 nitrifying bacteria at reduced concentration of oxygen. Appl. Environ. Microbiol. 40, 526^532. Kuai, L. and Verstraete, W. (1998) Ammonium removal by the oxygen-limited autotrophic nitrication-denitrication system. Appl. Environ. Microbiol. 64 (11), 4500^4506. Bock, E., Schmidt, I., Stuven, R. and Zart, D. (1995) Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonia or hydrogen as electron donors and nitrite as electron acceptor. Arch. Microbiol. 163, 16^20. Schmidt, I., Bock, E. and Jetten, M.S.M. (2001) Ammonia oxidation by Nitrosomonas eutropha with NO2 as oxidant is not inhibited by acetylene. Microbiology 147, 2247^2253. Mulder, A., van de Graaf, A.A., Robertson, L.A. and Kuenen, J.G. (1995) Anaerobic ammonium oxidation discovered in a denitrifying uidized bed reactor. FEMS Microbiol. Ecol. 16, 177^184. Jetten, M.S.M. (2001) New pathways for ammonia conversion in soil and aquatic systems. Plant Soil 230, 9^19. Broda, E. (1977) Two kinds of lithotrophs missing in nature. Z. Allg. Mikrobiol. 17, 491^493. Jetten, M.S.M., Strous, M., van de Pas-Schoonen, K.T., Schalk, J., van Dongen, L., van de Graaf, A.A., Logemann, S., Muyzer, G., van Loosdrecht, M.C.M. and Kuenen, J.G. (1999) The anaerobic oxidation of ammonium. FEMS Microbiol. Rev. 22, 421^437. Strous, M., Heijnen, J.J., Kuenen, J.G. and Jetten, M.S.M. (1998) The sequencing batch reactor as a powerful tool to study very slowly growing micro-organisms. Appl. Microbiol. Biotechnol. 50, 589^596. Strous, M., Fuerst, J., Kramer, E., Logemann, S., Muyzer, G., van de Pas, K., Webb, R., Kuenen, J.G. and Jetten, M.S.M. (1999) Missing lithotroph identied as new planctomycete. Nature 400, 446^449. Schmid, M., Twachtmann, U., Klein, M., Strous, M., Juretschko, S., Jetten, M., Metzger, J., Schleifer, K.H. and Wagner, M. (2000) Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst. Appl. Microbiol. 23, 93^106. Schlesner, H. and Stackebrandt, E. (1986) Assignment of the genera Planctomyces and Pirella to a new family Planctomycetaceae fam. nov. and description of the order Planctomycetales ord. nov. Syst. Appl. Microbiol. 8, 174^176. Fuerst, J.A. (1995) The planctomycetes : emerging models for microbial ecology, evolution and cell biology. Microbiology 141, 1493^ 1506. Griepenburg, U., Ward-Rainey, N., Mohamed, S., Schlesner, H., Marxen, H., Rainey, F.A., Stackebrandt, E. and Auling, G. (1999) Phylogenetic diversity, polyamine pattern and DNA base composition of members of the order Planctomycetales. Int. J. Syst. Bacteriol. 49, 689^696. Kuenen, J.G. and Jetten, M.S.M. (2001) Extraordinary anaerobic ammonium-oxidizing bacteria. ASM News 67, 456^463. Poulsen, L.K., Ballard, G. and Stahl, D.A. (1993) Use of rRNA uorescence in situ hybridization for measuring the activity of single cells in young and established biolms. Appl. Environ. Microbiol. 59, 1354^1360. Schmid, M., Schmitz-Esser, S., Jetten, M. and Wagner, M. (2001) 16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium oxidizing bacteria: implications for phylogeny and in situ detection. Environ. Microbiol. 3, 450^459. Oerther, D.B., Pernthaler, J., Schramm, A., Amann, R. and Raskin, L. (2000) Monitoring precursor 16S rRNAs of Acinetobacter spp. in

5. Conclusion Several new microbial pathways in the nitrogen cycle have been discovered. The planctomycete-like anammox bacteria converting ammonia and nitrite under anoxic conditions and the information about the exibility of the metabolism of aerobic nitriers add new possibilities to the nitrogen cycle. These two groups might even be natural partners in ecosystems with limited oxygen supply. Under these conditions aerobic ammonia oxidizers are able to oxidize ammonia to nitrite which will be consumed by anammox bacteria together with ammonia. As products of this cooperation mainly N2 and small amounts of nitrate are detectable [43]. When ammonia is the limiting substrate the anities of both groups of ammonia oxidizers might be decisive for the outcome of the competition. However, we are far from understanding the complexity of nitrogen conversion in detail. To gain deeper insight future studies might focus on regulation of nitrier metabolism, on community interactions, and on phylogenetic diversity of nitrogen converting microorganisms.
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