FEMS Microbiology Reviews 22 (1999) 421^437
The anaerobic oxidation of ammonium
Mike S.M. Jetten a; *, Marc Strous a , Katinka T. van de Pas-Schoonen a ,
Jos Schalk a , Udo G.J.M. van Dongen a , Astrid A. van de Graaf a ,
Susanne Logemann a , Gerard Muyzer 1;b , Mark C.M. van Loosdrecht a ,
J. Gijs Kuenen a
a
Kluyver Institute for Biotechnology, TU Delft, Julianalaan 67, NL-2628 BC Delft, The Netherlands
b
Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany
Received 26 June 1998; received in revised form 7 September 1998 ; accepted 7 September 1998
Abstract
From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist in
nature. `Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with
hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium
31
oxidation was about 2 nmol NH‡
(mg protein)31 using nitrogen dioxide as electron acceptor. This reaction, which may
4 min
involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for
growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot
plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism
31
was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH‡
(mg protein)31 was
4 min
determined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h31
(doubling time 11 days). 15 N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this
new process. A novel type of hydroxylamine oxidoreductase containing an unusual P468 cytochrome has been purified from the
Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia
occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater
treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously
assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner
for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for
future application in the removal of ammonium from wastewater with high ammonium concentrations. z 1999 Federation
of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Ammonium ; Nitrite ; Hydrazine; Hydroxylamine; Nitrosomonas; Oxygen; Wastewater; Nitrogen removal
* Corresponding author. Tel.: +31 (15) 2781193; Fax: +31 (15) 2782355; E-mail: [email protected]
1
Present address: Netherlands Institute for Sea Research NIOZ, Den Burg, Texel, The Netherlands.
0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 2 3 - 0
FEMSRE 624 25-1-99
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M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biological nature of the Anammox reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autotrophic growth during selective enrichment in continuous systems . . . . . . . .
Cultivation of Anammox biomass and determination of physiological parameters
Characterization of the enriched microorganisms . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Dominant cell type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Cytochrome spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Identi¢cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Aerobic versus anaerobic ammonium oxidation . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. In£uence of oxygen on anaerobic ammonium oxidation . . . . . . . . . . . . . . . .
6.2. Aerobic ammonium oxidizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Metabolic versatility of Nitrosomonas strains . . . . . . . . . . . . . . . . . . . . . . . .
7. Possible reaction mechanisms for Anammox . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Substrate spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9. Ecological habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Application of the Anammox process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. The combined SHARON-Anammox process . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The oxidation of ammonium has been investigated
mainly in aerobic or oxygen-limited systems. In
theory ammonium could also be used as an inorganic electron donor for denitri¢cation. The free energy for this reaction (Table 1) is nearly as favorable
as for the aerobic nitri¢cation process. It was on the
basis of these thermodynamic calculations that the
existence of two chemolithoautotrophic microorganisms capable of oxidizing ammonium to dinitrogen
gas was already predicted two decades ago [1]. The
actual discovery of such a process was only recently
Table 1
Gibbs free energy of several reactions involved in autotrophic
denitri¢cation [3,8]
Reaction equation
‡
2NO3
3 +5H2 +2H CN2 +6H2 O
ÿ
‡
+5HS
+3H
C4N2 +4H2 O+5SO23
8NO3
3
4
‡
‡
3NO3
3 +5NH4 C4N2 +9H2 O+2H
‡
NO3
2 +NH4 CN2 +2H2 O
3
‡
2O2 +NH‡
4 CNO3 +H2 O+2H
‡
C4N
+12H
O+8H
6O2 +8NH‡
2
2
4
vG³P
(kJ mol31 NH‡
4
or NO3
3 )
3560
3465
3297
3358
3349
3315
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422
422
423
424
425
425
425
425
427
427
428
430
431
433
433
434
435
435
436
described [2,3]. During experiments on a denitrifying
pilot plant of a multi-stage wastewater treatment system at Gist-Brocades (Delft, The Netherlands) it was
noted that ammonium disappeared from the reactor
e¥uent at the expense of nitrate with a concomitant
increase in dinitrogen gas production. A maximum
ammonium removal rate of 1.2 mmol l31 h31 was
observed. In continuous £ow experiments the nitrogen and redox balances showed that ammonium
really disappeared under anaerobic conditions, and
that for every mol of ammonium consumed 0.6 mol
of nitrate was required, resulting in the production
of 0.8 mol of dinitrogen gas. This newly discovered
process was named the Anammox (anaerobic ammonium oxidation) process.
2. Biological nature of the Anammox reaction
In a follow-up study, the biological nature of the
Anammox process was investigated in more detail
[4]. In anoxic batch experiments ammonium and nitrate were converted within 9 days of incubation
when intact seed material (Anammox biomass)
from the pilot plant was added. However, when the
seed material was subjected to Q-radiation or sterili-
FEMSRE 624 25-1-99
M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
zation at 121³C or when the seed material was omitted from the incubation no change in the concentration of nitrate and ammonium could be observed.
Furthermore the addition of various inhibitors (2,4dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone or HgCl2 ) to the incubations resulted in a
complete inhibition of the ammonium oxidation and
nitrate reduction (Table 2). In these experiments with
initial ammonium concentrations of 5 mM and higher, the rate of ammonium oxidation was proportional to the amount of biomass used. Taken together
these experiments strongly suggested that the anaerobic ammonium oxidation was a biological process
carried out by microorganisms. The speci¢c rate of
31
ammonium oxidation (0.08 nmol NH‡
(mg
4 min
31
dry weight) ) in these batch experiments was quite
31
low compared to rates (1.2 nmol NH‡
(mg
4 min
31
dry weight) ) obtained in the pilot plant. This indicated that in the batch experiments the conversion
was limited by di¡usion of the substrates to the biomass granules. Labeling experiments with 15 NH‡
4 in
combination with 14 NO3
3 showed an almost exclusive
production of 14ÿ15 N2 gas. This ¢nding did not agree
423
with the postulated overall reaction [3] (see Section
1) in which for every labeled ammonium, 0.6 nitrate
would react to form 0.8 dinitrogen gas (i.e. 75% of
the formed dinitrogen would be 15ÿ14 N2 and 25%
would be 15ÿ15 N2 ). Only if nitrite rather than nitrate
was assumed as the actual oxidizing agent the observed and calculated values would be in agreement
[4].
3. Autotrophic growth during selective enrichment in
continuous systems
Once it was realized that nitrite rather than nitrate
might be the electron acceptor of autotrophic denitri¢cation with ammonium as electron donor, a medium was composed for the selective enrichment of
the microorganisms responsible for the Anammox
process. This medium contained ammonium (5^
30 mM), nitrite (5^35 mM), bicarbonate (10 mM),
minerals and trace elements [5]. The phosphate concentration of the medium was kept below 0.5 mM
and the oxygen concentration below detection levels
Table 2
E¡ect of various treatments with stimulators and inhibitors on the anaerobic ammonium-oxidizing activity in batch experiments with
biomass from the Anammox pilot plant (adapted after [4,6,14])
Treatment inhibitor/stimulator
Mode of action
Concentration or
period tested
E¡ect
3
NH‡
4 ‡ NO2
No biomass
Sterilization at 121³C
Gamma irradiation
Penicillin V
Penicillin G
Bromoethane sulfonic acid
Na2 SO4
Na2 MoO4
Chloramphenicol
Hydrazine
Acetone
N-serve
Allylthiourea
Acetylene
activity test
none
denaturation
inactivation
inhibition of cell wall synthesis of bacteria
id.
inhibition of methanogenesis
stimulation of sulfate reduction
inhibition of sulfate reduction
inhibition of protein synthesis
inhibition of NH2 OH oxidation
solvent for N-serve
inhibition of nitri¢cation
inhibition of nitri¢cation
inhibition of nitri¢cation and
denitri¢cation
uncoupler
uncoupler
cell damage
oxidative stress
chelating agent
chelating agent
0^7 mM
0 mg l31
20^120 min
60 min
0^100 mg l31
0^1000 mg l31
0^20 mM
20 mM
20 mM
0^400 mg l31
0^3 mM
10 mM
0^50 mg l31
0^10 mM
6 mM
normal activity
no activity
no activity
no activtiy
none
none
none
none
none
none
activation
none
none
none
inhibition
0^400 mg l31
0^40 mg l31
0^300 mg l31
0^0.2 mM
6 1 mM
s 2 mM
inhibition
inhibition
inhibition
inhibition
none
inhibition
2,4-Dinitrophenol
Carbonyl cyanide m-chlorophenylhydrazone
HgCl2
Oxygen
Phosphate
Phosphate
FEMSRE 624 25-1-99
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M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
Fig. 1. Operation of a £uidized bed reactor for the enrichment and maintenance of anaerobic ammonium-oxidizing microorganisms. The
medium was composed of ammonium sulfate, sodium nitrite, NaHCO3 , minerals, trace elements [5,6]. The gray area represents the nitrite
and ammonium load into the reactor; the black area is the nitrite or ammonium load out of the reactor. The average removal percentage
(b) over 934 days was 99.5% for nitrite and 84.6% for ammonium.
( 6 1 WM) in order to avoid possible inhibitory e¡ects
(Table 2). Since the speci¢c rate of ammonium oxidation in batch experiments was considerably lower
than in perfusion systems a £uidized bed reactor was
chosen to perform the enrichment. Using biomass
from the original pilot plant as an inoculum, it was
possible to obtain stable enrichment cultures within
3^4 months of operation. In total more than 20 reactor runs have been carried out with synthetic medium, the longest (Fig. 1) lasting more than 27
months [5^7]. So far only two runs failed in the
enrichment mainly due to mechanical problems of
the setup. After enrichment with synthetic medium
the conversion rate in the reactor systems increased
33
from 0.4 kg NH‡
per day to about 3 kg NH‡
4 N m
4
33
N m per day. The maximum speci¢c activity of the
biomass in the £uidized bed reactor was about
31
25 nmol NH‡
(mg dry weight)31 . For every
4 min
mol of CO2 incorporated into biomass 24 mol of
ammonium had to be oxidized. When biomass
from the reactors was used in batch experiments
supplied with ammonium, nitrite and 14 CO2 , the cells
became rapidly labeled. The incorporation of label
was completely dependent on the combined presence
of both nitrite and ammonium. The ribulose bisphosphate carboxylase activity of cell extracts was
only 0.3 nmol CO2 min31 (mg dry weight)31 which
is 3-fold lower than expected on the basis of the
stoichiometry determined for ammonium and bicarbonate conversion (24:1). The estimated growth rate
in the £uidized bed systems was 0.001 h31 , which is
equivalent to 1 doubling time of about 29 days. The
main product of the reaction was dinitrogen gas,
but about 17% of the nitrite supplied could be
recovered as nitrate. The overall nitrogen balance
averaged over 15 runs showed a ratio of
1:1.31:0.22 for conversion of ammonium and nitrite
to the production of nitrate. In the £uidized bed
reactor no other intermediates like hydroxylamine,
hydrazine, NO or nitrous oxide could be detected.
The production of nitrate from nitrite was veri¢ed
with 15 N-NMR analysis [8]. Only when labeled nitrite was supplied to the cultures could formation of
15
15
NO3
N labeled
3 be observed. In the presence of
15
3
ammonium, no NO3 was ever observed [8]. The
function of this nitrate formation from nitrite is assumed to be the generation of reducing equivalents
necessary for the reduction of CO2 .
4. Cultivation of Anammox biomass and
determination of physiological parameters
Currently available microbiological techniques are
FEMSRE 624 25-1-99
M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
not designed very well to deal with very slowly growing microorganisms such as the Anammox culture.
In addition to the £uidized bed systems, a sequencing batch reactor (SBR) was applied and optimized
for the quantitative study of the microbial community that oxidized ammonium anaerobically [9]. The
SBR was a powerful experimental setup in which the
biomass was retained very e¤ciently ( s 90%). Furthermore a homogeneous distribution of substrates,
products and biomass aggregates over the reactor
was achieved, and the reactor has been in operation
reliably for more than 2 years under substrate-limiting conditions. Several important physiological parameters ([9] M. Strous, personal communication)
such as the biomass yield (0.066 þ 0.01 C mol (mol
ammonium)31 ), the maximum speci¢c ammonium
consumption rate (45 þ 5 nmol min31 (mg
protein)31 ) and the maximum speci¢c growth rate
(0.0027 h31 , doubling time 11 days) could now be
determined more accurately than with the £uidized
bed reactors. The temperature range for Anammox
was 20^43³C (with an optimum at 40³C). The Anammox process functioned well at pH 6.7^8.3 (with an
optimum at pH 8). Under optimal conditions the
maximum speci¢c ammonium oxidation rate was
about 55 nmol min31 (mg protein)31 . The a¤nity
for the substrates ammonium and nitrite was very
high (a¤nity constants 9 5 WM) (M. Strous, personal
communication). The Anammox process was inhibited by nitrite at nitrite concentrations higher than 20
mM but lower nitrite concentrations ( s 10 mM)
were already suboptimal. When nitrite was present
at high concentrations for a longer period (12 h),
Anammox activity was completely lost. In addition,
the persisting stable and strongly selective conditions
of the SBR led to a high degree of enrichment (74%
of the desired dominant peculiar microorganisms, see
Section 5).
5. Characterization of the enriched microorganisms
5.1. Dominant cell type
The dominant microorganism of the enrichment
cultures was a Gram-negative light-breaking coccoid
cell (Fig. 2A,B) which showed an unusual irregular
morphology under the electron microscope (Fig. 2C)
425
when ¢xed with 2.5% glutaraldehyde in 20 mM
K2 HPO4 /KH2 PO4 bu¡er pH 7.4. Once the unusual
morphology of the cells was recognized, an estimate
of the enrichment from the original culture could be
made by counting the cells in a large number of thin
sections. After 177 days of enrichment 64% of all
cells counted (7317 out of 11 433 total) were of the
described type. This was a four-fold increase (16%;
1632 out of 10 200 total) compared to the numbers
found in the biomass from the pilot plant. Together
with the increase in cell numbers of this peculiar
organism an increase in the percentage of ether-like
lipids was observed. The amount of ester lipids typical for most Bacteria remained more or less constant [5]. The presence of the ether lipids seems to
be con¢ned to most ancient microorganisms such as
the Archaea or very deep-branching Bacteria like
Thermotoga and Aquifex. A detailed knowledge of
the exact structure and composition of the lipids of
the dominant cell type would be most helpful to ¢nd
the taxonomic a¤liation of these cells [5].
5.2. Cytochrome spectra
During the enrichment on synthetic medium, the
color of the biomass changed from brown to deep
red. Visible spectra of cells and cell extracts of the
enriched culture showed a pronounced increase in
the signal for cytochromes of the c type. Spectra of
cells at 77 K revealed the absence of a-type, b-type
and d1 -type cytochromes. Very interestingly, during
increase in Anammox activity of the biomass, gradually an increased signal was observed at 468 nm. In
Fig. 3 a typical spectrum of an Anammox cell extract
with the 468-nm feature is shown. This absorption
peak at 468 nm disappeared irreversibly after treatment with carbon monoxide. A similar signal has
been observed in aerobic ammonium-oxidizing bacteria at 463 nm [10^13]. This signal was attributed to
one of the hemes present in the enzyme hydroxylamine oxidoreductase and is mostly referred to as
cytochrome P460.
5.3. Identi¢cation
So far many isolation methods including serial dilution, £oating ¢lters, and plating have been used to
obtain the responsible microorganisms in pure cul-
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M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
427
Fig. 2. A: Micrograph of a biomass aggregate from an Anammox enrichment culture. The dominant coccoid cell is present in packages
and microcolonies. B: Micrograph of the dominant coccoid cell present in the Anammox enrichment cultures. Preparation was obtained
after sedimentation of suspended material from a £uidized bed reactor. C: Electron micrograph of suspended Anammox biomass ¢xed
with 2.5% glutaraldehyde in 20 mM K2 HPO4 /KH2 PO4 bu¡er pH 7.4. The micrograph was taken at the Department of Electron Microscopy (I. Keizer, K. Sjollema, M. Veenhuis), State University of Groningen, The Netherlands.
ture. None of the isolates thus obtained is able to
perform the Anammox reaction, but many of the
isolates are novel denitrifying oligotrophic (proteo)bacteria. In addition to classical microbial techniques, the Anammox community was analyzed using
modern molecular biological methods. The genomic
DNA was extracted and ampli¢ed via PCR using
(eu)bacterial primers 27f-BamHI (5P-CACGGATCCAGAGTTTGATMTGGCTTCAG-3P), and 1492rHindIII (5P-TGTAAGCTTACGGYTACCTTGTTACGACT-3P). The PCR products were cloned, and
396 out of the more than 4000 clones obtained were
screened. One dominant (28%) clone belonging to
the Cytophaga/Flexibacter phylum was identi¢ed.
However, in situ analysis with £uorescent probes
speci¢c for the Cytophaga phylum did not con¢rm
the molecular identity of the dominant organism as a
Cytophaga.
6. Aerobic versus anaerobic ammonium oxidation
The presence of a P460 -like signal in the enriched
Anammox biomass, and the recent reports on the
metabolic versatility of aerobic ammonium oxidizers
initiated a more detailed investigation. The studies
were concentrated on three issues: the in£uence of
oxygen on the Anammox process, the number of
aerobic ammonium oxidizers present in the Anammox enrichment cultures and the (anaerobic) capabilities of `classical' nitri¢ers of the Nitrosomonas
type.
6.1. In£uence of oxygen on anaerobic ammonium
oxidation
The in£uence of oxygen on the Anammox process
was investigated in both batch and continuous sys-
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428
M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
Fig. 3. Cytochrome spectrum of cell extract from an Anammox enrichment culture. Dashed line, oxidized spectrum; solid line, spectrum
after reduction with dithionite. Inset shows a close-up between 440 and 600 nm. The arrow indicates the typical peak at 468 nm.
tems. Initial batch experiments showed that oxygen
completely inhibited the Anammox activity when it
was deliberately introduced into the enrichment cultures [5,14]. In a follow-up study an intermittently
oxic (2 h) and anoxic (2 h) reactor system was
used to study the reversibility of the oxygen inhibition for 20 days [15]. From these studies it became
clear that ammonium was not oxidized in the oxic
periods, but that the Anammox activity in the anoxic
periods remained constant throughout the experiment, indicating that the inhibitory e¡ect of oxygen
was indeed reversible. The sensitivity of the Anammox enrichment culture to oxygen was further
investigated under various sub-oxic conditions. In
four consecutive experiments, the oxygen tension
was decreased stepwise from 2 to 0% of air
saturation (Fig. 4). No ammonium was oxidized
in the presence of 0.5, 1, or 2% of air. Only when
all the air was removed from the reactor by
vigorously £ushing with argon gas, the conversion
of ammonium and nitrite resumed, thus indicating
that the Anammox activity in these enrichment
cultures is only possible under strict anoxic conditions.
6.2. Aerobic ammonium oxidizers
The second question which was addressed in these
studies concerned the number of aerobic ammoniumoxidizing bacteria present in the Anammox enrichment cultures. Using standard, aerobic most probable number (MPN) methods, the number of nitri¢ers was estimated to be 9 þ 5U103 cells of
ammonium oxidizers per milliliter of biomass sample. Electron micrographs of the MPN cultures
showed the characteristic cytoplasmic membrane
structures reported for several aerobic ammoniumoxidizing bacteria [16]. The consistent presence of
these aerobic nitri¢ers in the Anammox biomass con¢rmed that they can survive very long periods of
anaerobiosis as was previously shown by Abeliovich
[17]. Furthermore, it was possible to enrich such organisms in a repeated fed-batch reactor when oxygen
was continuously supplied at 50^80% of air saturation [15]. The enriched aerobically nitrifying community grew exponentially with a doubling time of
1.2 days. Interestingly, not all of the 27 mM of ammonium supplied could be recovered as nitrite. Since
nitrite was not further oxidized to nitrate, the re-
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429
Fig. 4. The Anammox activity at four di¡erent air saturations (A 2%, B 1%, C 0.5%, and D 0%). Only when all oxygen was removed
from the incubation by £ushing with argon gas the disappearance of ammonium (b) and nitrite (F) could be observed [15].
mainder of the nitrogen might have been lost as gaseous nitrogen compounds (NO, N2 O or N2 ) during
aerobic denitri¢cation. After enrichment of the nitri¢ers, also this culture was subjected to the same alternating 2 h oxic/2 h anoxic regime to verify if these
nitri¢ers were capable of an anaerobic conversion of
ammonium or nitrite. During 20 days, the fate of the
supplied 30 mM ammonium was followed and it was
observed that only in the aerobic periods oxidation
of ammonium to nitrite occurred. This indicated that
this community of aerobic ammonium-oxidizing bacteria was not able to use nitrite as an electron acceptor in this case.
This is in contrast to observations made with several pure cultures of di¡erent Nitrosomonas strains.
Poth showed that a new Nitrosomonas isolate was
able to produce dinitrogen gas under anaerobic conditions [18,19], while Abeliovich and Vonshak demonstrated the reduction of nitrite with pyruvate as
electron donor by N. europaea [20].
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Table 3
Rates of anaerobic oxidation (nmol min31 (mg protein)31 ) of ammonium, hydroxylamine and hydrazine by various cultures in batch experiments
Culture
Compounds tested
NO3
2
conversion rate
NH2 OH/NH‡
4 /N2 H4
conversion rate
Products
Reference
N.
N.
N.
N.
N.
NH2 OH+NO3
2
3
NH‡
4 ‡ NO2
H2 +NO3
2
3
NH‡
4 ‡ NO2
‡
NH4 ‡ NO3
2
2
2
7
61
0.9
3
3
not applicable
61
1.1
N2 O
N2 O
N2 O, N2
N2 O
NO, N2 O
[23]
[23]
[16]
[16]
[21]
3
NH‡
4 ‡ NO2
NH2 OH
N2 H4 +NO3
2
3
NH‡
4 ‡ NO2
12
n/a
13
68
9
12
11
55
N2
N2
NH‡
4 , N2
N2
[5]
[8]
[8,24]
[9], M. Strous,
personal communication
europaea
europaea
eutropha
eutropha
eutropha
Anammox
Anammox
Anammox
Anammox
6.3. Metabolic versatility of Nitrosomonas strains
More recently substantial N losses have been reported for both mixed and pure cultures of N. eutropha grown under oxygen limitation [16,21,22], and
for pure cultures of N. europaea in anoxic batch experiments [23]. When molecular hydrogen was used
as an electron donor for nitrite reduction, growth of
N. eutropha was stoichiometrically coupled to nitrite
reduction with dinitrogen gas and nitrous oxide as
end products. In mixed cultures of N. eutropha and
Enterobacter aerogenes 2.2 mM of ammonium and
nitrite were consumed during 44 days of incubation,
but no cell growth was observed [16]. In a follow-up
study the rate of anaerobic ammonium oxidation by
N. eutropha could be estimated at 0.08 nmol NH‡
4
min31 (mg protein)31 which is equivalent to about
31
0.2 nmol NH‡
(mg dry weight)31 . However,
4 min
when the nitrogen atmosphere of the incubations
was supplemented with 25 ppm nitrogen dioxide,
31
the rate increased 10-fold to 2.2 nmol NH‡
4 min
31
(mg protein) [21]. It was estimated that 40^60% of
the formed nitrite (and NO) was denitri¢ed to dinitrogen gas while N2 O and hydroxylamine were detected as intermediates. The source of oxygen for the
oxidation of ammonia under these anoxic conditions
remained unknown, but could theoretically be derived from either NO, NO2 or nitrite. Very recently
it was shown that N. eutropha also exhibited denitrifying capabilities in the presence of NO2 when the
dissolved oxygen concentration was maintained at
3^4 mg l31 [22]. In these experiments with complete
biomass retention 50% of the produced nitrite was
aerobically denitri¢ed to dinitrogen gas. NO gas was
much less e¤cient in stimulating this aerobic denitri¢cation than NO2 and became toxic above 25 ppm.
Furthermore, an eight-fold increased aerobic nitri¢-
Fig. 5. Concentration pro¢le of ammonium (b), nitrite (F), hydroxylamine (R) and hydrazine (8) during anaerobic batch experiments with an Anammox culture supplemented with 3 mM
hydroxylamine [8].
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431
Table 4
Comparison of the properties of the hydroxylamine oxidoreductase (HAO) enzyme isolated from Anammox (J. Schalk, personal communication) and Nitrosomonas europaea [12]
Molecular mass
Subunit
Composition
Ratio 410/280
Heme
Active center
Vmax
Km
pH optimum
pI
N-terminus
HAO of Anammox
HAO of Nitrosomonas
150 kDa
60^95 kDa
K2 ^K3
4.5
22 þ 4/150 kDa
P468
21 U mg31
26 WM
8
5.5
blocked
125^175 kDa
63 kDa
K2 ^K3
3.3
24/K3
P463
75 U mg31
not reported
8
5.3
DISTV
cation rate and higher cell numbers were observed
when the air was supplemented with 25^50 ppm
NO2 . In Table 3 a summary is presented of the reported rates of anaerobic ammonium oxidation in
various experiments with Nitrosomonas and/or
Anammox cultures. From this table it is evident
that the speci¢c rates for anaerobic ammonium oxidation of the classical nitri¢ers are 25-fold lower
than the rates observed in the Anammox process
studied in Delft. Furthermore, aerobic ammonium
oxidizers prefer to use oxygen as the terminal electron acceptor, whereas this compound completely
inhibits the Anammox process. Taken together these
examples showed that further research to elucidate
the role of nitrogen oxides during (an)aerobic ammonium oxidation is necessary.
7. Possible reaction mechanisms for Anammox
The possible metabolic pathway for anaerobic ammonium oxidation was investigated using 15 N labeling experiments. These experiments showed that
ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor [8].
The hydroxylamine itself is most likely derived
from nitrite. In batch experiments with excess hydroxylamine and ammonium, a transient accumulation of hydrazine was observed (Fig. 5). The conversion of hydrazine to dinitrogen gas is postulated as
the reaction generating the electron equivalents for
the reduction of nitrite to hydroxylamine. Whether
the reduction of nitrite and the oxidation of hydra-
Fig. 6. Possible reaction mechanisms and cellular localization of
the enzyme systems involved in anaerobic ammonium oxidation.
A: Ammonium and hydroxylamine are converted to hydrazine
by a membrane-bound enzyme complex, hydrazine is oxidized in
the periplasm to dinitrogen gas, nitrite is reduced to hydroxylamine at the cytoplasmic site of the same enzyme complex responsible for hydrazine oxidation with an internal electron transport. B: Ammonium and hydroxylamine are converted to
hydrazine by a membrane-bound enzyme complex, hydrazine is
oxidized in the periplasm to dinitrogen gas, the generated electrons are transferred via an electron transport chain to nitrite reducing enzyme in the cytoplasm.
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zine occur at di¡erent sites of the same enzyme (Fig.
6A) or the reactions are catalyzed by di¡erent enzyme systems connected via an electron transport
chain (Fig. 6B) remains to be investigated. The occurrence of hydrazine as an intermediate in microbial
nitrogen metabolism is rare [24]. Hydrazine has been
proposed as an enzyme-bound intermediate in the
nitrogenase reaction [25]. Furthermore, the puri¢ed
hydroxylamine oxidoreductase (HAO) of N. europaea is capable of catalyzing the conversion of hydrazine to dinitrogen gas [12]. The ¢nding of high
HAO activity in cell extracts of the Anammox culture indicated that a similar enzyme might be operative in the Anammox process. Indeed very recently
a novel type of HAO was puri¢ed from the Anammox community via anion exchange and gel ¢ltration chromatography (J. Schalk, personal communication). Native PAGE showed that the Anammox
enzyme had a smaller molecular mass than the enzyme of Nitrosomonas. Furthermore, the amino acid
sequence of several HAO peptide fragments was
unique, without any homologue in the databases.
Similar to the Nitrosomonas hydroxylamine oxidoreductase, the enzyme from Anammox contained several c-type cytochromes. The special spectroscopic
P460-like feature was found at 468 nm in the Anammox enzyme. The enzyme was able to catalyze the
oxidation of both hydroxylamine and hydrazine.
Although hydroxylamine was the preferred substrate
the rate of hydrazine oxidation in cell extracts (150
nmol min31 (mg protein)31 ) was high enough to
sustain a growth rate of 0.003 h31 . In Table 4
some properties of the two HAO enzymes are summarized (J. Schalk, personal communication).
Fig. 7. Concentration pro¢le of ammonium (b) and nitrite (F) in
the absence of methane in the head space, and of ammonium
(a) and nitrite (E) in the presence of 50% methane in the argon/
CO2 head space during anoxic batch experiments with Anammox
biomass.
The formation of hydroxylamine via an ammonium monooxygenase seems highly improbable, since
the Anammox reaction is strongly but reversibly inhibited by oxygen. An alternative mechanism for the
formation of hydroxylamine might be the incomplete
reduction of nitrite by a cytochrome c-type nitrite
reductase. However, it will be very di¤cult to obtain
direct evidence for this mechanism in Anammox.
Hydroxylamine is the compound most rapidly metabolized by Anammox, and a selective inhibitor
has not yet been discovered.
Table 5
Possible reaction equations of anaerobic ammonium oxidation via NO or HNO as intermediates, adapted after [12,14]
NO as intermediate
NO+NH3 +3H‡ +3e3
N2 H4
‡
3
NO3
2 +2H +e
CN2 H4 +H2 O
CN2 +4H‡ +4e3
CNO+H2 O
‡
NH3 +NO3
2 +H
CN2 +2H2 O
HNO as intermediate
HNO+NH3
N2 H2
‡
3
NO3
2 +2H +2e
CN2 H2 +H2 O
CN2 +2H‡ +2e3
CHNO+OH3
NH3 +NO3
2
CN2 +H2 O+OH3
(ammonia monooxygenase-like enzyme)
(hydroxylamine oxidoreductase-like enzyme)
(nitrite reductase)
(ammonia monooxygenase-like enzyme)
(hydroxylamine oxidoreductase-like enzyme)
(nitrite reductase)
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Fig. 8. The absence of methane conversion by Anammox biomass at two di¡erent methane concentrations in the head space.
Closed triangles (R, 380 Wmol methane) and diamonds (8, 195
Wmol methane) represent incubations with Anammox biomass in
the presence of 300 Wmol nitrite as electron acceptor. The closed
circles represents a control incubation of Anammox biomass with
280 Wmol ammonium (b), and 300 Wmol nitrite as electron acceptor. In the control all nitrite was converted, in the incubations
with methane the nitrite was not converted.
433
indicated that the enzyme responsible for anaerobic
ammonium conversion is di¡erent from the aerobic
ammonia or methane monooxygenases. In longer experiments it could also be shown that methane itself
was not converted by the Anammox biomass (Fig.
8). In addition to methane also hydrogen was tested
in batch incubations (Fig. 9). The addition of hydrogen to the argon/CO2 head space showed a clear
stimulation of the anaerobic ammonium oxidation
in short-term experiments. However, hydrogen could
not replace ammonium as electron donor in these
experiments. Addition of various organic substances
(pyruvate, methanol, ethanol, alanine, glucose, casamino acids) in short-term batch experiments led to a
severe inhibition of the Anammox activity. Thus the
substrate spectrum seems to be restricted to ammonium, nitrite and the intermediates hydrazine and
hydroxylamine. However, supplementation with
1 mM hydrazine could not sustain growth of the
Anammox culture for longer periods [24].
9. Ecological habitats
The discovery of the Anammox process in a deni-
A possible role of NO or HNO in (an)aerobic
ammonium oxidation was proposed by Hooper [12]
to be a condensation of NO or HNO and ammonia
on an enzyme related to the ammonium monooxygenase family (Table 5). The formed hydrazine or
imine could thereafter be converted by the enzyme
hydroxylamine oxidoreductase into dinitrogen gas
and the reducing equivalents required to combine
NO or HNO and ammonia or to reduce nitrite to
NO.
8. Substrate spectrum
Aerobic ammonium and methane oxidizers are
able to catalyze both the oxidation of ammonium
and methane [26] albeit at di¡erent rates. The ability
of the Anammox culture to use methane or other
substrates was tested in batch experiments. In Fig.
7 it is shown that addition of methane to the argon/
CO2 head space of the incubations did not lead to an
inhibition of ammonium and nitrite conversion. This
Fig. 9. Concentration pro¢le of ammonium (b) and nitrite (F) in
the presence of 95% hydrogen and 5% CO2 gas in the head
space, and of ammonium (a) and nitrite (E) in the presence of
95% argon and 5% CO2 gas in head space during anoxic batch
experiments with Anammox biomass.
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Table 6
Overview of the parameters of an Anammox £uidized bed reactor [7,38] and a SHARON reactor [38] both fed with sludge digester e¥uent. The nitrite for the Anammox process was supplied separately
Ammonium load
Nitrite load
Nitrogen load
NH‡
4 N e¥uent
NO3
2 N e¥uent
E¤ciency NH‡
4 N
Removal
E¤ciency NO3
2 N
Removal
Sludge load
a
SHARON
Anammox
0.63^1.0a
not applicable
0.63^1.0
199
469
76^90
0.24^1.34
0.22^1.29
0.46^2.63
27 þ 85
3þ3
88 þ 9
33
31
kg NH‡
4 N m reactor day
3
33
kg NO2 N m reactor day31
kg Ntot m33 reactor day31
mg N l31
mg N l31
%
n/a
99 þ 2
%
10.3
0.05^0.26
kg Ntot kg31 SS per day
This value is linearly proportional to the in£uent concentration.
trifying pilot plant has raised the question as to
where else such organisms would occur in nature.
Already in 1932 it was reported that dinitrogen gas
was generated via an unknown mechanism during
fermentation in the sediments of Lake Mendota
(USA) [27]. Also in sediments of Lake Kizakiko (Japan) indications were found for the direct formation
of dinitrogen gas from ammonium [28]. Very recently these observations were con¢rmed in studies
with freshwater sediments [29]. One prerequisite for
the occurrence of anaerobic ammonium oxidation
via an Anammox mechanism would be the simultaneous presence of both ammonium and nitrite (or
nitrate) and the absence of oxygen. The nitrite could
be formed either in ecosystems in which oxygen (diffusion) limits nitri¢cation or in places with a limited
supply of electron donor (sul¢de or organic substances) for denitri¢cation of nitrate. The oxic/anoxic
interface of many sediments would thus be an ideal
habitat for anaerobic ammonium-oxidizing microorganisms. Micro-electrode studies have revealed overlapping pro¢les of nitrate and ammonium in a strati¢ed zone of the Black Sea [30,31] indicating that a
habitat for Anammox really exists. More recently
after the development of nitrite microsensors, also
overlapping pro¢les of nitrite and ammonium have
been reported in oxygen-limited nitrifying activated
sludge £ocs [32^34]. Indeed man-made ecosystems
like wastewater treatment plants could create a habitat for the Anammox organisms. The abundant supply of ammonium via the wastewater in combination
with a limited oxygen availability would provide
conditions in which both ammonium and nitrite
could occur. High nitrogen losses (70^90%) in the
form of dinitrogen gas have been reported in two
rotating biological contractor systems [35^37] treating land¢ll leachate with about 200^400 mg ammonium per liter. Comparison of the microorganisms in
these systems with the Delft Anammox culture
would give more insight into the biodiversity of the
anaerobic ammonium oxidation.
10. Application of the Anammox process
In a recent study [7,38] the feasibility of the Anammox process for the removal of ammonium from
sludge digester e¥uents was evaluated. The results
of this study showed that the Anammox biomass
was not negatively a¡ected by the digester e¥uent.
The pH (7.0^8.5) and temperature (30^37³C) optima
for the process were well within the range of the
values found in digester e¥uents. Experiments with
a laboratory-scale (2-l) £uidized bed reactor showed
that the Anammox biomass was capable of removing
ammonium and (externally added) nitrite e¤ciently
from the sludge digester e¥uent (Table 6). The nitrogen load of the Anammox £uidized bed reactor
could be increased from 0.46 kg Ntot m33 reactor per
day to 2.6 kg Ntot m33 reactor per day. Due to the
nitrite limitation, the maximum capacity was not
reached. The nitrogen conversion rate during the experiment with sludge digester e¥uents increased
from 0.05 kg Ntot kg31 SS per day to 0.26 kg Ntot
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M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
kg31 SS per day. The Anammox sludge biomass removed 88% of the ammonium and 99% of the nitrite
from the sludge digester e¥uent (Table 6). In these
studies nitrite was supplied from a concentrated
stock solution. However, for application in real
wastewater practice, a suitable system for biological
nitrite production has to be developed. One such
system is the SHARON (single reactor high activity
ammonium removal over nitrite) process [38].
This SHARON process is ideally suited to remove
nitrogen from waste streams with a high ammonium
concentration ( s 0.5 g N l31 ). The SHARON process is performed in a single, stirred tank reactor without any sludge retention. At temperatures above
25³C it is possible to e¡ectively outcompete the nitrite oxidizers. This results in a stable nitri¢cation
with nitrite as end-product [38]. When combined
with the Anammox process only 50% of the ammonium needs to be converted to nitrite. This implies
that no extra addition of base is necessary, since
most of the wastewater resulting from anaerobic digestion will contain enough alkalinity (in the form of
bicarbonate) to compensate for the acid production
if only 50% of the ammonium needs to be oxidized.
The SHARON process has been extensively tested at
the laboratory scale for the treatment of sludge digester e¥uents (Table 6) and is currently under construction at two Dutch wastewater treatment plants.
11. The combined SHARON-Anammox process
The combination of the Anammox process and
SHARON process has been tested in our laboratory
Table 7
Nitrogen balances in the combined SHARON-Anammox system
SHARON
In£uent
NH‡
4 584 (100%)
NO3
2 61
NO3
3 61
N2 Oa 6 1
N2 a 6 1
Anammox
E¥uent/In£uent
E¥uent
267(46%)
227 (39%)
64 (11%)
4
61
29 (5%)
1.4
83 (14%)
61
476b (82%)
Results are mg N l31 , percentages are given in parentheses.
a
Concentration relative to the in£uent £ow.
b
Determined as the di¡erence between dissolved and gaseous nitrogen compounds.
435
Fig. 10. Ammonium removal from sludge digester e¥uent with
the combined SHARON-Anammox system. Ammonium (8) or
nitrite (F) load in the e¥uent of the SHARON reactor is used
as the ammonium or nitrite load into the Anammox reactor. The
ammonium or nitrite load in the e¥uent of the Anammox is represented by open diamonds and open squares, respectively. The
pH value in the Anammox reactor was stable at 7.8 [38].
using sludge digester e¥uent (Fig. 10). The
SHARON reactor was operated without pH control
with a total nitrogen load of about 0.8 kg N m33 per
day [38]. The ammonium present in the sludge
digester e¥uent was converted to nitrite and a small
amount of nitrate (11%) (Table 7). The nitrate formation was due to bio¢lm wall growth, which was
not always regularly removed. In large-scale applications this will be signi¢cantly lower because of the
larger volume to surface ratio. In this way an ammonium-nitrite mixture suitable for the Anammox
process was generated. The e¥uent of the SHARON
reactor was used as in£uent for the Anammox £uidized bed reactor. In the nitrite-limited Anammox reactor all nitrite was removed, the surplus ammonium
remained. During the test period the overall ammonium removal e¤ciency was 83%. In Table 7 the
nitrogen balances of the two systems are summarized. The optimization and application of the combination of these two processes on pilot plant and full
scale remain challenges towards implementations in
a future wastewater treatment plant.
Acknowledgments
The research on anaerobic ammonium oxidation
FEMSRE 624 25-1-99
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M.S.M. Jetten et al. / FEMS Microbiology Reviews 22 (1999) 421^437
was ¢nancially supported by the Foundation for Applied Sciences (STW), the Foundation of Applied
Water Research (STOWA), the Netherlands Foundation for Life Sciences (NWO-SLW), the Royal
Netherlands Academy of Arts and Sciences
(KNAW), Gist-Brocades, DSM, and Grontmij consultants. The contributions of various co-workers
and students over the years are gratefully acknowledged.
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