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FEMS Microbiology Ecology 39 (2002) 175^181
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MiniReview
Aerobic and anaerobic ammonia oxidizing bacteria ^
competitors or natural partners?
Ingo Schmidt
b
a;
*, 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
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(anammox) and the finding of an anoxic metabolism by ‘classical’ Nitrosomonas-like organisms showed that this is no longer valid.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. 9 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Nitri¢cation; Anammox ; ‘Candidatus Brocadia anammoxidans’ ; NOx cycle; Nitrosomonas ; Aerobic ammonia oxidation; Anaerobic ammonia
oxidation
1. Introduction
Nitri¢cation is an important part of the biological nitrogen cycle. Microorganisms involved in nitri¢cation are
characterized as lithotrophic ammonia and nitrite oxidizing bacteria and heterotrophic nitri¢ers (not discussed in
this review). Lithotrophic nitri¢ers 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 bio¢lms on the surfaces of
substrata [5]. Nitri¢ers 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 nitri¢ers 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 : [email protected] (I. Schmidt).
2.1. Molecular identity
Although Broda [21] predicted the existence of chemo-
0168-6496 / 01 / $22.00 9 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 nitri¢ers under anoxic conditions, the ¢rst experimental con¢rmation 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 e¥uent
with a concomitant increase of dinitrogen gas production.
The microbial nature of the process was veri¢ed, and nitrite was shown to be the preferred electron acceptor [22].
Hydroxylamine and hydrazine were identi¢ed 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 e⁄cient 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 puri¢ed from enrichment cultures by density gradient centrifugation [24].
DNA extracted from the puri¢ed cells was used as a template for PCR ampli¢cation 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 speci¢c 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 bio¢lm 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 re£ecting the relationships of Candidatus ‘Kuenenia stuttgartiensis’ and Candidatus ‘Brocadia anammoxidans’ to organisms a⁄liated to the order Planctomycetales. The tree is based on results of maximum likelihood analyses from di¡erent 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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177
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 .
3
3
þ
3
NHþ
4 þ 1:32 NO2 þ 0:066 HCO3 þ 0:13 H ! 0:26 NO3 þ
1:02 N 2 þ 0:066 CH 2 O0:5 N 0:15 þ 2:03 H 2 O
ð1Þ
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 43‡C [39]. Under optimal conditions, the speci¢c activity is about 3.6 mmol (g protein)31 h31 , the biomass
yield about 0.066 C-mol (mol ammonium)31 , and the speci¢c 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 Fe3þ , Mn4þ , 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 e¡ect 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-arti¢cial ecosystem, a fresh water swamp in Uganda [42]. Oxic/anoxic
interfaces are abundant in nature, for example in bio¢lms
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 di¡erent 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].
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 speci¢c ampli¢cation 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
con¢ned to two monophyletic lineages within the Proteobacteria. Nitrosococcus oceanus is a⁄liated 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 nitri¢ers can be detected even in
anoxic habitats.
NH3 þ N2 O4 þ 2 Hþ þ 2 e3 ! NH2 OH þ H2 O þ 2 NO
ð2Þ
NH2 OH þ H2 O ! HNO2 þ 4 Hþ þ 4 e3
ð3Þ
NH3 þ N2 O4 ! HNO2 þ 2 NO þ 2 Hþ þ 2 e3
ð4Þ
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 þ 3 Hþ þ 3 e3 ! 0:5 N2 þ 2 H2 O
ð5Þ
There are only a few di¡erences 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 signi¢cant
¢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 in£uence 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-
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:
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 di¡erent 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
denitri¢cation by ammonia oxidizing bacteria [55,56]. Additionally, the formation of dinitrogen was observed
[17,57]. Furthermore, Nitrosomonas is capable of anoxic
denitri¢cation with molecular hydrogen [17] or simple organic compounds [14] serving as electron donors.
179
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.
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. Nitri¢cation 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
signi¢cantly reduced when NOx was added. Evidence is
given that the cells generate NO for the NOx cycle via
denitri¢cation when external NOx is not available [59].
Nitrogenous oxides have a signi¢cant promoting e¡ect
on pure cultures of N. eutropha [59,60]. Their addition
resulted in a pronounced increase in nitri¢cation rate, speci¢c activity of ammonia oxidation, growth rate, maximum cell density, and aerobic denitri¢cation capacity.
Maximum cell numbers amounted to 2U1010 Nitrosomonas cells ml31 . Furthermore, about 50% of the nitrite produced was aerobically denitri¢ed 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 þ N2 O4 þ 2 Hþ þ 2 e3 ! NH2 OH þ 2 NO þ H2 O
ð6Þ
2NO þ O2 ! 2 NO2 ðN2 O4 Þ
ð7Þ
NH3 þ O2 þ 2 Hþ þ 2 e3 ! NH2 OH þ H2 O
ð8Þ
The sum of Eqs. 6 and 7, given in Eq. 8, was already
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 di¡erent. 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 di¡erent 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
nitri¢cation 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
con¢rmed 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 speci¢c signal molecules
between ammonia oxidizers [59].
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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’ nitri¢ers 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 a⁄nities 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 nitri¢er metabolism,
on community interactions, and on phylogenetic diversity
of nitrogen converting microorganisms.
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