Published Ahead of Print 15 February 2013.
2013, 79(8):2807. DOI:
Appl. Environ. Microbiol.
Jetten and Boran Kartal
Kleerebezem, Mark van Loosdrecht, Jans Kruit, Mike S. M.
Ziye Hu, Tommaso Lotti, Merle de Kreuk, Robbert
Nitritation-Anammox Bioreactor at Low
Nitrogen Removal by a
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Nitrogen Removal by a Nitritation-Anammox Bioreactor at Low
Ziye Hu,aTommaso Lotti,bMerle de Kreuk,c* Robbert Kleerebezem,bMark van Loosdrecht,bJans Kruit,dMike S. M. Jetten,a,b
Department of Microbiology, IWWR, Radboud University Nijmegen, Nijmegen, The Netherlandsa; Department of Biotechnology, Delft University of Technology, Delft,
The Netherlandsb; Waterschap Hollandse Delta, Ridderkerk, The Netherlandsc; Paques BV, Balk, The Netherlandsd
life, on the receiving bodies. Generally, carbonaceous waste is re-
by nitrogen removal systems. Conventionally, the removal of ni-
trogen (ammonium) is accomplished by the combination of ni-
trification and denitrification processes. Both of these are energy
consuming and are associated with high costs. Moreover, these
processes have an additional environmental impact due to high
biomass production and greenhouse gas (CO2, N2O, etc.) emis-
sion, which promote global warming.
ammonium and nitrite directly to dinitrogen gas (N2) under an-
oxic conditions. Since they were first detected in a denitrifying
pilot plant by Mulder et al. in 1995 (1), anammox bacteria have
been found in various oxygen-limited natural (2–4) and man-
made ecosystems. The application of the anammox process in
wastewater treatment results in significant energy reduction
(60%) and greenhouse gas emission (90%) compared to those of
traditional biological nitrogen removal processes (5–7). In full-
scale nitritation-anammox wastewater treatment plants, ammo-
supplied ammonium to nitrite under O2limitation, and in turn,
nitrite, together with the remaining ammonium, is converted to
for the treatment of warm and high-strength wastewaters, such as
digester effluents and anaerobically treated industrial effluents
A more sustainable municipal wastewater treatment can be
achieved with a first step in which available organic carbonaceous
compounds are concentrated and converted to CH4in an anaer-
itrogen removal from wastewater treatment is necessary be-
municipal wastewater has a lower temperature (?10 to 15°C,
apart from tropical and subtropical regions) and a relatively low
ammonium concentration (12). This may lead to lower specific
activities and growth rates for both anammox bacteria and AOB.
Indeed, it was reported that the activities of anammox bacteria
and AOB both decreased at 15 to 20°C (13, 14) and that partial
nitrification was difficult to achieve in winter because of the vary-
ing temperature of municipal wastewater (15). Nevertheless, sev-
eral studies showed that nitrogen removal at a lower temperature
by an anammox process can work (14, 16, 17); still, in none of
these studies was it possible to maintain a stable anammox-AOB
culture (nitritation-anammox) at temperatures lower than 20°C.
On the other hand, in natural ecosystems, such as Northern Eu-
ropean soils and marine sediments, anammox bacteria thrive un-
der much colder temperatures and very low ammonium concen-
trations (?M range) (18, 19). Therefore, it should be possible to
adapt a nitritation-anammox bioreactor to low ammonium con-
Received 24 December 2012 Accepted 12 February 2013
Published ahead of print 15 February 2013
Address correspondence to Boran Kartal, firstname.lastname@example.org.
*Present address: Merle de Kreuk, Department of Water Management, Delft
University of Technology, Delft, The Netherlands.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
April 2013 Volume 79 Number 8 Applied and Environmental Microbiologyp. 2807–2812aem.asm.org
on June 12, 2014 by guest
centrations to treat cold municipal wastewater using appropriate
tor (SBR) was inoculated with anammox biomass from an SBR
operating at 30°C. Oxygen was supplied gradually to allow the
growth of AOB. The enriched nitritation-anammox biomass was
adapted to 12°C. Synthetic wastewater simulating pretreated mu-
nicipal wastewater was used as influent for the bioreactor. Activi-
ties of anammox, AOB, and aerobic nitrite oxidizers (NOB) were
monitored by offline batch tests, and the microbial community
composition was investigated by molecular analyses, including
fluorescence in situ hybridization (FISH) and clone libraries. The
responses of specific activity of each functional community to
temperature change and low ammonium concentrations were
MATERIALS AND METHODS
Reactor setup and operation. Two sequencing batch reactors (SBR;
working volume, 5 liters) were used for the cultivation of anaerobic am-
monium-oxidizing bacteria (anammox) and nitritation-anammox bio-
grow anammox biomass at different temperatures as follows. The anam-
mox SBR was stirred at 200 rpm with a six-bladed turbine stirrer. Each
drawing of the liquid. During each filling period, 1 liter of mineral me-
supplied to the anammox SBR. To maintain anoxic conditions, the reac-
5%; 10 ml · min?1). CO2present in the supplied gas and bicarbonate in
in the reactor at ?7.3. The temperature of the reactor was maintained at
30°C with a water bath. The temperature of the anammox SBR was de-
activity tests were performed to determine the anammox activity as de-
scribed below (“Activity tests”).
When the anammox SBR described above was operated at 30°C, one
liter of this culture (70 to 80% enriched) was used as seed sludge to inoc-
ulate another SBR to enrich nitritation-anammox biomass as follows. At
the time of the inoculation, the temperature was decreased in one step
from 30°C to 25°C. The nitritation-anammox SBR was operated as de-
scribed above with respect to SBR cycle, pH, and anoxia. During each
filling period of the SBR cycle, 1 liter of mineral medium (21) containing
420 mg N/liter (30 mM) nitrite and 588 mg N/liter (42 mM) ammonium
was supplied for 93 days. Oxygen (7.54 mg/day) was introduced to the
mimic municipal wastewater, ammonium and nitrite concentrations
nium and nitrite in the influent were 70 mg N/liter (5 mM; day 191) and
temperature of the reactor was stepwise decreased to 12°C (Fig. 1). Bio-
mass (1.5 ml) was collected every 2 days for DNA extraction and every
week for FISH analysis. When the system was operated at 12°C, over a
period of 15 days (at days 330, 338, and 345), triplicate gas samples (100
?l) were taken for N2O measurements from both SBRs.
mox, AOB, and NOB) was tested at days 13, 113, 137, and 268, which
represent different operational stages (different oxygen supply and tem-
perature). For anammox activity tests, 10 ml biomass was taken directly
from the anammox SBR and the nitritation-anammox SBR. The samples
2 mM (14 to 28 mg N) nitrite and ammonium and adjustment of the pH
natively applying vacuum and supplying Ar/CO2(95%/5%) at least 7
60 min (depending on actual activity) for ammonium and nitrite mea-
For AOB and NOB activity tests, 10 ml biomass from the nitritation-
anammox SBR was transferred to a 50-ml Erlenmeyer flask under atmo-
spheric conditions. Biomass used for the NOB activity test was first
washed 3 times with HEPES buffer (20 mM, pH 7.4) to remove all am-
the anammox activity tests.
Analytical methods. Collected liquid samples were centrifuged (5
min, 10,000 ? g), and the supernatants were kept at ?20°C until further
the Biuret method as previously described (23). N2O was measured with
an Agilent 6890 Series GC (Agilent Technologies) equipped with a Pora-
pack Q column and an electron capture detector (ECD).
nitritation-anammox SBR as described previously (24) with probes spe-
cific for Kuenenia and Brocadia-like anammox bacteria (AMX820 ),
Kuenenia stuttgartiensis (KST1273 ), AOB (NEU653 ), and NOB
(NTSPA0712  and NIT1035 ). DAPI (4=,6-diamidino-2-phe-
nylindole) was used to stain the whole community DNA.
DNA extraction, PCR amplification, cloning, and phylogenetic
analyses. Genomic DNA was extracted from 1 ml of centrifuged sample
stored at ?20°C until further analyses. DNA concentrations were deter-
mined by the NanoDrop 1100 spectrophotometer (Thermo Scientific).
the primer combination pla46F (28) and 1529R (30). For quantitative
PCR (qPCR), the primer combinations hzsA526F/hzsA1829R (31) and
amoA-1F/amoA-2R (32) were used for anammox bacteria and AOB, re-
spectively, using the MyiQ single-color real-time PCR detection system
Drop 1100 spectrophotometer and then serially diluted in 10-fold steps
before qPCR was performed. For all PCR amplifications, a 25-?l reaction
mixture containing 12.5 ?l GoTaq Green master mix (Promega Benelux
template DNA was used. PCR products were ligated to pGEM-T easy
FIG 1 Operation of the laboratory-scale nitritation-anammox SBR under
different conditions. Filled and empty circles indicate ammonium concentra-
tions in the influent and effluent, respectively, and filled triangles indicate
nitrite concentrations in the influent. Nitrite concentrations in the effluent
were always below the detection limit. Empty triangles indicate nitrate con-
centrations in the effluent.
Hu et al.
aem.asm.org Applied and Environmental Microbiology
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vector (Promega Benelux BV, Leiden, the Netherlands) according to the
JET plasmid miniprep kit (Fermentas GMBH, St. Leon-Rot, Germany),
and the insert was sequenced by the M13 Forward primer (5= GTAAAAC
GACGGCCAGT 3=). Phylogenetic trees were constructed using MEGA 5
Enrichment of the aerobic ammonium-oxidizing bacteria. The
anammox biomass (?80% enriched) from the sequencing batch
reactor (SBR) operated at 30°C was used as an inoculum for the
nitritation-anammox SBR. After inoculation, the bioreactor was
operated at 25°C and anaerobically for 2 weeks with 420 mg N/li-
ter (30 mM) nitrite and 588 mg N/liter (42 mM) ammonium as
period, anammox bacteria were responsible for all ammonium
to the activity of AOB. At day 34, Nitrosomonas-like microorgan-
isms were detected by FISH, and aerobic ammonium-oxidizing
activity was detectable, indicating that an AOB community had
already developed within 20 days. On day 119, the concentration
of ammonium was decreased to 210 mg N/liter (15 mM) and
nitrite was no longer supplied to the reactor. After this point, the
nitrite required for the anammox reaction was completely pro-
duced by the AOB. To mimic pretreated municipal nitrogenous
wastewater, the ammonium concentration was lowered to 70 mg
N/liter (5 mM) on day 191 (Fig. 1) and was kept at this value for
the rest of the experimental period. Ammonium supplied to the
nitritation-anammox SBR was partially oxidized by AOB to ni-
tween days 125 and 136), the temperature of the reactor could be
gradually lowered from 25°C to 12°C without nitrite accumula-
tion. On day 150, the temperature of the reactor was lowered to
viated this accumulation.
Changes of community composition in response to temper-
ature and oxygen. The results of FISH analysis of the biomass
from the nitritation-anammox SBR showed a clear increase of
the start of O2addition, still only anammox bacteria were able to
the abundance of AOB was apparently below the detection limit
(10,000 cells/ml, ?1% of the microbial population). With in-
creasing O2supply, the abundance of AOB increased to about
50% of the total population (day 135) (see Fig. S1 in the supple-
mental material) and anammox bacteria and AOB comprised ap-
shown). Clone libraries (see Fig. S2 in the supplemental material)
and FISH analyses revealed that after the introduction of oxygen,
the dominant anammox species in the bioreactor was a “Candi-
out the operation of the nitritation-anammox SBR. It was not
possible to detect NOB with the most general probes during the
Functional gene abundance of anammox and AOB. Anam-
mox bacteria and AOB in the nitritation-anammox SBR were
quantified by real-time qPCR performed on the hzsA and amoA
genes, respectively. At day 35, 20 days after the introduction of
oxygen and when the temperature was 25°C, copy numbers of
hzsA and amoA genes were ?7 ? 109copies · ml?1and ?4 ? 107
of the total population. The anammox copy numbers corre-
sponded to an in situ anammox activity of 10 fmol N · cell?1·
day?1. An increase in amoA gene copy numbers, which was con-
gruent with the FISH results, was observed subsequently. At day
copy numbers of hzsA and amoA genes were ?2 ? 107copies ·
in situ anammox and AOB activities were 10.5 and 24 fmol N ·
cell?1· day?1, respectively.
The effect of temperature and oxygen on activities of differ-
ent trophic groups. In the 100 days after the introduction of O2,
there was a decrease in the observed anammox activity from 16.6
to 12 nmol N · mg total protein?1· min?1in the nitritation-
anammox SBR. This decrease was most probably due to the fact
that the AOB were now comprising a larger part of the total bio-
mass and consuming part of the ammonium. AOB activity dou-
total protein?1· min?1at 12°C. NOB activity was undetectable
during the whole experimental period, which indicated that all
substrates were consumed by anammox and AOB.
When the nitritation-anammox SBR was operated at 12°C for
tion to low temperature (Table 1). To investigate the effect of
temperature on anammox activity, biomass from the 12°C nitri-
tation-anammox SBR and the anammox SBR was sampled and
incubated at different temperatures. The optimum temperature
for the anammox biomass from the nitritation-anammox SBR
TABLE 1 Activities of anammox, AOB, and NOB at different operation stages
aValues are expressed in nmol N · mg protein?1· min?1. ND, not detectable.
Nitritation-Anammox Bioreactors at 12°C
April 2013 Volume 79 Number 8aem.asm.org 2809
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enriched at 12°C was 25°C, 10°C lower than that for the biomass
originating from the anammox SBR operated at 30°C. Interest-
tein) of anammox bacteria grown at 30°C and 12°C were almost
identical (Fig. 2). Moreover, cold-adapted biomass still oxidized
ammonium at a rate of 5 nmol N · mg protein?1· min?1at 12°C
(Fig. 2), which was a high enough rate to remove all nitrogen
supplied to the system. The AOB from the cold-adapted reactor
had the maximum specific activity (activity per AOB protein) at
Anammox bacteria oxidize a part of their substrate nitrite to
nitrate for reducing equivalents necessary for cell carbon fixation;
therefore, when anammox bacteria are growing, nitrate produc-
tion is observed. With the decreasing temperature, there was also
a decrease in nitrate production by anammox bacteria in the ni-
tritation-anammox SBR. In the 68 days before the temperature
was lowered, the average ratio of nitrate produced to ammonium
consumed by the anammox bacteria was approximately 0.18,
decreased to 0.04, suggesting that the anammox bacteria had a
lower yield and/or higher maintenance activity.
emission from the nitritation-anammox SBR at 12°C was ?2.4%
(? 0.1%) of total N removed (Fig. 3).
In this study, first the temperature of the bioreactor of an existing
out adverse effects, indicating that the anammox bacteria had a
sufficient overcapacity. Then a coculture of aerobic ammonium-
oxidizing bacteria (AOB) and anammox bacteria was enriched in
the same reactor by introducing a limiting amount of oxygen to
the bioreactor. After establishing a stable coculture consisting of
perature of the nitritation-anammox SBR was decreased from 25
to 12°C. It was possible to decrease the temperature of the biore-
actor within 10 days without nitrite accumulation, and this de-
crease did not result in a change in the dominant anammox spe-
cies in the bioreactor. Conversely, when the temperature of the
reactor was lowered to 9°C, there was a gradual (from day 150 to
temperature back to 12°C resulted in the consumption of the ac-
cumulated nitrite. This indicates that even though it was not pos-
activity was reversible. Such a nitrite accumulation may be be-
cause AOB have a higher activity than anammox bacteria at 9°C
(Fig. 2). Nevertheless, it cannot currently be ruled out that with a
slower decrease of temperature or by using a different type of
biomass (e.g., granular), complete nitrite conversion can be
reached at temperatures lower than 12°C. Considering the long
doubling time of anammox bacteria (10 to 25 days) (34), the
acclimation period was very short, indicating that sludge from
full-scale bioreactors operating at 30°C may be used to seed new
bioreactors designed to remove nitrogen from pretreated low-
temperature municipal wastewaters without an extended adapta-
Growth of the anammox bacteria is always associated with ni-
nitrite to nitrate as the ultimate source for the electrons that are
used for cell carbon fixation. The stoichiometric ratio of nitrate
production to ammonium consumption for the anammox bacte-
ria is 1:0.26. When the temperature of the nitritation-anammox
SBR was decreased from 25°C to 12°C, this ratio decreased from
0.18 (average of the first 68 days) to 0.04 (the average of the last
and/or much higher cell maintenance activity at a lower temper-
ature. This phenomenon may be the reason why the decrease in
temperature resulted in a 100-fold decrease in the copy numbers
of the anammox bacteria. Nevertheless, the decrease in tempera-
in situ activity rates of aerobic and anaerobic ammonium-oxidiz-
that these groups of microorganisms had an overcapacity for the
of microorganisms in the nitritation-anammox SBR, although
FIG 2 Effect of long-term enrichment at 30°C (filled circles, solid line) and
12°C (empty circles, dotted line) on the maximum specific activity of the
temperatures are also depicted (filled triangles, dashed line). Protein concen-
trations on the y axis indicate anammox or AOB protein only.
FIG 3 N2O emission from the nitritation-anammox SBR (12°C) (A) com-
pared to that from anammox cultures enriched at 30°C (B) and 15°C (C)
and a full-scale nitritation-anammox wastewater treatment plant (32 to
33°C) (46) (D).
Hu et al.
aem.asm.org Applied and Environmental Microbiology
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sufficient to remove more than 90% (average of the last 100 days,
92%) of the supplied ammonium, indicating that the full-scale
and low ammonium concentration may be feasible.
There was a clear optimum temperature shift (35 to 25°C) in
range (10 to 35°C). This indicated that the activity loss caused by
low temperature was reversible and that the common seasonal
changes in temperature during full-scale applications would not
affect the stability of the treatment system.
Currently, nitritation-anammox reactors are operated at tem-
peratures higher than 30°C (10, 11), and previous studies on the
physiology of the anammox bacteria reported that the optimum
growth temperature for anammox bacteria is around 30 to 35°C
(see reference 35 for a review). Moreover, most described AOB
have an optimal temperature of around 28°C (36). On the other
hand, some studies indicated that nitrite-oxidizing bacteria were
to 17°C (36) and that at 10°C to 15°C, NOB had a higher activity
anammox bacteria below 20 to 25°C, depending on their affinity
for nitrite and O2, they could take up the limiting O2and nitrite
before AOB and anammox bacteria, respectively. In turn, this
would lead to nitrate production and, eventually, the collapse of
the system. Nevertheless, in the natural ecosystems in which aer-
obic and anaerobic ammonium-oxidizing bacteria thrive (for ex-
ample, oxygen minimum zones), the temperature is well below
10°C, indicating that both clades of microorganisms are able to
compete with (or outcompete) NOB (38, 39). This was also the
case in our nitritation-anammox enrichment culture under oxy-
material). Even an extended enrichment at 12°C (132 days) did
not facilitate the growth of NOB. Our results showed that anam-
mox bacteria directly consumed nitrite produced by AOB, and
affinity to nitrite than anammox bacteria (40, 41). In our labora-
formation. Nevertheless, balancing aeration with various ammo-
nium loads may prove difficult to achieve in full-scale applica-
tions. Therefore, a thorough study at the pilot scale would be
necessary to determine the optimal process conditions and con-
trol parameters that would lead to a stable full-scale operation.
encing the emission of N2O from nitritation-anammox bioreac-
tors, most of which is attributed to the activity of AOB (42–45).
Our results were completely in line with this observation: there
was a negligible (0.02%) amount of N2O produced in the anam-
the nitritation-anammox SBR, ?2.4% of removed nitrogen was
detected as N2O (Fig. 3). This value was remarkably similar to the
(?2.6% of removed nitrogen). Nevertheless, it should be noted
that the laboratory-scale nitritation-anammox bioreactor in this
study was operated under controlled and stable conditions. On
the other hand, in a full-scale application, many parameters, in-
cluding wastewater quality, efficiency of the previous treatment
steps, and temperature, fluctuate and the results obtained here
cannot be used to directly estimate N2O emissions from full-scale
nitrogen removal systems.
In this study, we present the proof of principle for the applica-
tion of the nitritation-anammox process for nitrogen removal
from pretreated municipal wastewater. It was possible to adapt a
nitritation-anammox bioreactor to low temperature and low am-
monium load very rapidly. Moreover, the lab-scale nitritation-
anammox bioreactor was operated for over 300 days without ni-
trite accumulation and was able to remove over 90% of the
supplied nitrogen at temperatures as low as 12°C. Nevertheless,
of the application of such a process at full scale. We believe that
these should focus especially on determining process control pa-
rameters for the optimal operation of nitritation-anammox bio-
reactors under variable wastewater conditions and the degree of
greenhouse gas emissions (e.g., N2O) from these systems.
This research was supported by a KRW grant (European Union Water
Framework Directive, grant number 09035) and STOWA (Foundation
for Applied Water Research, The Netherlands). Z.H. and M.S.M.J. were
supported by ERC 232937, and B.K. was supported by the Netherlands
Organization for Scientific Research (VENI grant 863.11.003).
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