aerobic and anaerobic ammonia oxidizing bacteria ^

8/6/2019 Aerobic and Anaerobic Ammonia Oxidizing Bacteria ^

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MiniReview

Aerobic and anaerobic ammonia oxidizing bacteria ^

competitors or natural partners?

Ingo Schmidt 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 Netherlandsb Delft University of Technology, Kluyver Laboratory for Biotechnology, Department of Microbiology and Enzymology, Julianalaan 67,

2628 BC Delft, The Netherlandsc 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(anammox) and the finding of an anoxic metabolism by classical Nitrosomonas-like organisms showed that this is no longer valid.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 nitro-

gen cycle. Microorganisms involved in nitrication are

characterized as lithotrophic ammonia and nitrite oxidiz-

ing bacteria and heterotrophic nitriers (not discussed in

this review). Lithotrophic nitriers are all placed in the

family Nitrobacteraceae [1], although they are not neces-

sarily related phylogenetically. Chemolithoautotrophic ni-

trifying bacteria have been found in many ecosystems such

as fresh water, salt water, sewage systems, soils, and on/inrocks as well as in masonry [2,3]. Growth under subopti-

mal conditions might be possible by ureolytic activity, ag-

gregate 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 as-

sumed. 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 N2O4 as oxi-

dant for ammonia oxidation under both oxic and anoxicconditions [18]. Furthermore, a new group of anaerobic

nitrite-dependent ammonia oxidizers (anammox) were dis-

covered [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)

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 16 8 - 6 4 9 6 ( 0 1 ) 0 0 2 0 8 -2

* Corresponding author.

Tel.: +31 (24) 3652568; Fax: +31 (24) 3652830.

E-mail address: [email protected] (I. Schmidt).

FEMS Microbiology Ecology 39 (2002) 175^181

www.fems-microbiology.org


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lithoautotrophic bacteria capable of anaerobic ammonium

oxidation and Abeliovich [14] reported high cell concen-

trations of nitriers under anoxic conditions, the rst ex-

perimental 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 euentwith a concomitant increase of dinitrogen gas production.

The microbial nature of the process was veried, and ni-

trite was shown to be the preferred electron acceptor [22].

Hydroxylamine and hydrazine were identied as impor-

tant 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 reten-

tion 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 iso-late the microorganisms with classical methods failed.

Therefore, the bacterium was physically puried from en-

richment cultures by density gradient centrifugation [24].

DNA extracted from the puried cells was used as a tem-

plate 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 anaer-

obic ammonium oxidizing planctomycete-like bacterium

was named Candidatus Brocadia anammoxidans. The

16S rDNA sequence information was used to design spe-

cic oligonucleotide probes for application in uorescence

in situ hybridization (FISH) and to survey the presence ofB. anammoxidans and related anammox bacteria in several

wastewater treatment systems [25]. Indeed, B. anammoxi-

dans and the closely related Candidatus Kuenenia stutt-

gartiensis 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 environ-

mentally derived 16S rDNA sequences [20,28] strongly in-

dicate further planctomycete lineages [29], including the

anammox bacteria (Fig. 1). In fact, the newly found bac-

terium 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 FISHprobes showed the dominance of these bacteria in ecosys-

tems 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 indi-

cator of physiological activity [32]. Therefore, the inter-

genic spacer regions (ISR) between the 16S rRNA and

23S rRNA, as part of the precursor rRNA, of B. anam-moxidans and K. stuttgartiensis were sequenced. Subse-

Fig. 1. 16S rDNA-based phylogenetic dendrogram reecting the relationships of Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anam-

moxidans 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.

I. Schmidt et al./ FEMS Microbiology Ecology 39 (2001) 175^181176


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quently, ISR-targeted oligonucleotide probes were con-

structed and applied by FISH. Inhibition experiments

with B. anammoxidans revealed a good correlation be-

tween 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-grow-

ing bacteria [31].

2.3. Ecophysiology

The ultrastructure of B. anammoxidans has many fea-

tures in common with previously described planctomy-

cetes. These microorganisms have a proteinaceous cell

wall lacking peptidoglycan and are thus insensitive to

ampicillin. The chromosome is separated from the sur-

rounding cytoplasm by a single or double membrane. In

B. anammoxidans an additional compartment bounded by

a single membrane [33], free from ribosomes and chromo-

some, was observed. This peculiar organelle made upmore 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 en-

zyme was localized in this middle compartment, which

was named anammoxosome [33]. Interestingly, B. anam-

moxidans [33] as well as aerobic ammonia oxidizers such

as Nitrosomonas [1] develop internal membrane systems.

Whether such a membrane system is bioenergetically nec-

essary for ammonia oxidation is still the subject of inves-

tigation since both key enzymes of Nitrosomonas are ob-

viously not localized in the intracytoplasmic membrane

(ICM) system. According to the peptide structure theAMO was described as a membrane-bound enzyme [35],

and recent studies [36] indicated a localization in the cy-

toplasmic membrane. The HAO is localized in the peri-

plasm [37].

To unravel the metabolic pathway for anaerobic ammo-

nium oxidation in B. anammoxidans, series of 15N-labeling

experiments were conducted. It could be shown that am-

monium 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. Ac-cording to the working hypothesis, the oxidation of hydra-

zine to dinitrogen gas is supposed to generate four elec-

trons 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 ni-

trate is assumed to be the generation of reducing equiva-

lents necessary for the reduction of CO2.

NH4 1:32 NO3

2 0:066 HCO3

3 0:13 H ! 0:26 NO33

1:02 N2 0:066 CH2O0:5N0:15 2:03 H2O 1

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 ac-tivity is about 3.6 mmol (g protein)31 h31, the biomass

yield about 0.066 C-mol (mol ammonium)31, and the spe-

cic growth rate about 0.0027 h31. Recent studies showed

that K. stuttgartiensis is in many ways similar to B. anam-

moxidans [40]. K. stuttgartiensis cells have the same overall

cell structure and also produce hydrazine from exoge-

nously 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 pa-

rameters are necessary. For example the anammox bacte-ria are very sensitive to oxygen and nitrite. Oxygen con-

centrations as low as 2 WM and nitrite concentrations

between 5 and 10 mM inhibit the anammox activity com-

pletely, but reversibly [22].

3. Ecology of anammox

In various ecosystems B. anammoxidans will be depen-

dent on the activity of aerobic ammonia oxidizing bacteria

under oxygen-limited conditions, e.g., at the oxic/anoxic

interface. Anammox biomass has already been detectedin wastewater treatment plants in The Netherlands, Ger-

many, Switzerland, UK, Australia, and Japan [42]. Re-

cently anammox cells were detected in a non-articial eco-

system, 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 am-

monia oxidizers would oxidize ammonium to nitrite and

keep the oxygen concentration low, while B. anammoxi-

dans 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

Fig. 2. Proposed model for the anaerobic ammonia oxidation (anam-

mox) of Brocadia-like microorganisms. HH: hydrazine hydrolase; HZO:

hydrazine oxidizing enzyme; NR: nitrite reducing enzyme.

I. Schmidt et al./ FEMS Microbiology Ecology 39 (2001) 175^181 177


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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 ox-

idizers as has been documented before [46,47]. It seemslikely that under these conditions anaerobic and aerobic

ammonia oxidizers form a quite stable community. The

cooperation of aerobic and anaerobic ammonium oxidiz-

ing bacteria is not only relevant for wastewater treatment

[45,48], but might play an important role in natural envi-

ronments at the oxic/anoxic interface. Further interactions

under anoxic conditions between both groups of ammonia

oxidizers seem to be likely since an anoxic, NO2-depen-

dent 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 lithoauto-

trophic organisms using carbon dioxide as the main car-

bon source. Several species reveal extensive ICM systems.

Recently, molecular tools to detect the presence of ammo-

nia oxidizing bacteria in the environment have been sup-

plemented by PCR primers for specic amplication of the

ammonia monooxygenase structural gene amoA [3]. Envi-ronmental 16S rRNA and amoA libraries have extended

the knowledge on the natural diversity of ammonia oxidiz-

ing bacteria [49]. Comparative 16S rRNA sequence anal-

yses revealed that members of this physiological group are

conned to two monophyletic lineages within the Proteo-

bacteria. Nitrosococcus oceanus is aliated with the Q-sub-

class 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 anaero-

bic ammonia oxidation by Nitrosomonas [51]. These results

indicate a complex role of nitrogen oxides (NO and NO2)

in the metabolism of aerobic ammonia oxidizers. Nitro-

somonas 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 tetrox-

ide, nitrite, and nitric oxide:

NH3 N2O4 2 H 2 e3 ! NH2OH H2O 2 NO

2

NH2OH H2O ! HNO2 4 H 4 e3 3

NH3

N2

O4

! HNO2

2 NO 2 H 2 e3 4

Hydroxylamine and nitric oxide are formed in this reac-

tion. While nitric oxide is not further metabolized, hydrox-

ylamine 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 H2O 5

There are only a few dierences between the anaerobic,

NO2-dependent and the aerobic, O2-dependent [53] am-

monia oxidation by Nitrosomonas. Instead of O2 in the

course of aerobic ammonia oxidation, N2O4 is used as

electron acceptor and NO, an additional product, is re-leased 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 ammo-

nia oxidation with NO2 (N2O4) as oxidant was not af-

fected by acetylene [18]. N. eutropha cells treated with

acetylene oxidized ammonia even under oxic conditions

if NO2 was available. Ammonia oxidation was not detect-

able in the absence of NO2. One of the most signicant

ndings is that the 27-kDa polypeptide of the AMO was

not labeled with [14C]acetylene during anoxic NO2-depen-

dent ammonia oxidation. When oxygen was added, thelabeling of this polypeptide with [14C]acetylene started im-

mediately. 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. N2O4 is the oxidant for the am-

monia oxidation.



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ence of the oxidizing agent N2O4. NO is produced in stoi-

chiometric amounts (Fig. 3). Only when NO2 is available

under anoxic conditions, ammonia is oxidized and hydrox-

ylamine occurs as an intermediate while NO is formed as

an end product. Hydroxylamine is further oxidized to ni-

trite [52].

Under anoxic conditions nitrite serves as a terminalelectron acceptor. In the absence of ammonia Nitrosomo-

nas is capable of using dierent substrates as electron do-

nor. During hydroxylamine oxidation by ammonia oxidiz-

ers, 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]. Ad-

ditionally, the formation of dinitrogen was observed

[17,57]. Furthermore, Nitrosomonas is capable of anoxic

denitrication with molecular hydrogen [17] or simple or-

ganic compounds [14] serving as electron donors.

4.3. Aerobic ammonia oxidation

NOx also plays an important role in the aerobic metab-

olism of nitrifying microorganisms. Nitrosomonas-like or-

ganisms 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 eecton pure cultures of N. eutropha [59,60]. Their addition

resulted in a pronounced increase in nitrication rate, spe-

cic activity of ammonia oxidation, growth rate, maxi-

mum cell density, and aerobic denitrication capacity.

Maximum cell numbers amounted to 2U1010 Nitrosomo-

nas cells ml31. Furthermore, about 50% of the nitrite pro-

duced was aerobically denitried to dinitrogen when nitro-

gen oxides were present.

In the presence of O2, the produced NO can be (re)oxi-

dized to NO2 [18]. Therefore, only small amounts of NO

are detectable in the gas phase of Nitrosomonas cell sus-

pensions. According to the model (Fig. 4, Eq. 6), N2O4 isthe oxidizing agent under oxic conditions. Hydroxylamine

and NO are produced as intermediates. While hydroxyl-

amine is further oxidized to nitrite, NO is (re)oxidized to

NO2 (N2O4) (Eq. 7):

NH3 N2O4 2 H 2 e3 ! NH2OH 2 NO H2O

6

2NO O2 ! 2 NO2 N2O4 7

NH3 O2 2 H 2 e3 ! NH2OH H2O 8

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

described earlier as the reaction of aerobic ammonia oxi-

dation [53], but is in complete agreement with the new

hypothetical model. The total consumption rates (ammo-

nia, oxygen) and production rates (hydroxylamine as in-

termediate) are the same, but the mechanism of the reac-

tion is quite dierent. Since detectable NOx concentrations

were small, nitrogen oxides seem to cycle in the cell (pos-

sibly enzyme-bound). Therefore, the total amount of NOxper cells is expected to be low. This hypothetical model

(Fig. 4) is in good accordance with the described mecha-

nisms 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 ofhydroxylamine still originates from molecular oxygen, but

is incorporated via NO2 [18].

In control experiments dierent species of ammonia oxi-

dizers were tested (e.g., Nitrosomonas europaea, Nitrosolo-

bus multiformis) [61]. All species were able to oxidize am-

monia under anoxic conditions with NO2 as oxidant (Fig.

3) and the aerobic ammonia oxidation activity was in-

creased in the presence of NO or NO2 (Fig. 4).

4.4. Ecological evidence of NOx

The ecological evidence of nitrogen oxides (NOx) fornitrication 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 activ-

ity by denitrifying Nitrosomonas cells (hydrogen as elec-

tron donor) is regulated via the availability of NO2. Sec-

ond, in contrast to homoserine lactones, which function as

signal molecules between many bacteria [62], nitrogen ox-

ides seem to function as very specic signal molecules

between ammonia oxidizers [59].

Fig. 4. NOx cycle. Hypothetical model of the ammonia oxidation by

Nitrosomonas. According to this model, N2O4 is the oxidant for the am-

monia oxidation. Under oxic conditions oxygen is used to re-oxidize

NO to NO2 (N2O4). Hydroxylamine is oxidized to nitrite.



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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 nitriers add new possibilitiesto 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 prod-

ucts 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 oxidiz-

ers 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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