73

［Japanese Journal of Water Treatment Biology Vol.52  No.3  73－83  2016］

Startup of Lab-scale Anammox Reactors

Seeded with Activated Sludge
at Ambient Temperature
GIRI PARK1, NAMI TESHIMA2, YUKI NAKAYAMA2, MASASHI TAKEKAWA1, 2,
SATOSHI SODA1, MICHIHIKO IKE1, KAZUICHI ISAKA3, and KENJI FURUKAWA2
1
Graduate School of Engineering, Osaka University/2－1 Yamada-oka, Suita, Osaka 565－0871, Japan
2
Department of Civil and Environmental Engineering, Faculty of Engineering, Kumamoto University
/2－39－1 Kurokami, Kumamoto 860－8555, Japan
3
Matsudo Research Center, Hitachi, Ltd., Infrastructure System Company
/537 Kami-hongo, Matsudo, Chiba 271－0064, Japan

Abstract
The optimal temperature for anaerobic ammonium oxidation (anammox) is generally
30－37℃. This temperature range, which is higher than usual wastewater temperature,
implies that the anammox reactor requires heat energy for efficient nitrogen removal.
Therefore, the anammox process which is applicable at ambient temperature (20－25℃) is
needed to develop more versatile and sustainable nitrogen removal technologies. This
study was conducted to start-up lab-scale anammox reactors packed with a nonwoven
fabric material at 20℃ using various inoculum sources. Reactor 1 was inoculated with
activated sludge sampled in Kumamoto, a temperate region of Japan. Reactor 2 was
inoculated with activated sludge obtained from Hokkaido, a subarctic region. Reactor 3
was inoculated with groundwater microorganisms sampled from Kumamoto. At 800 days
from the start-up of the reactors, Reactors 1 and 2 achieved high nitrogen removal rates
(NRR) of 0.64 kg－N/m3/day at 20℃ with the typical anammox reaction ratio. Although the
nitrogen removal was unstable, the NRR increased to 0.38 kg－N/m3/day in Reactor 3 after
700 days. The nitrogen reaction ratio in Reactor 3 indicated the simultaneous occurrence
of anammox and nitrification. Results suggest that anammox bacteria, which can grow at
ambient temperature, are ubiquitous in the environment.

Key words: anaerobic ammonium oxidation, enrichment, ambient temperature

However, the anammox process generally

INTRODUCTION
Anaerobic ammonium oxidation (anammox)
is a biologically mediated reaction in which
ammonium ( NH4 －N ) is oxidized to nitrogen
gas using nitrite ( NO2 －N ) as the electron
acceptor. Nitrogen removal processes using
anammox1)－3) can provide important benefits
compared to conventional processes: less
oxygen demand, external carbon supply, and
sludge production. Recently, the anammox
process has been implemented for treating
wastewater generated by some industries4).
requires an additional heating system to
maintain 30－37℃ temperatures for efficient
nitrogen removal5)－9), which lowers its energy
efficiency.
Consequently, the application of the
anammox process to wastewater treatment at
ambient temperature (≤  25℃) has attracted
attention worldwide. Actually, anammox
bacteria are reportedly present in the
environment with diverse ranges of temper­
ature: 10－43℃9). Furthermore, many reports
have described adaptability to lower tem­
74 Japanese J. Wat. Treat. Biol. Vol.52 No.3

perature of the anammox bacteria enriched mesophilic anammox bacteria to lower

in mesophilic conditions6),10)－19). However, most
reports of the literature have described
that lower temperatures of around 20℃
severely damaged the specific anammox
activity6),10),12),13),18)－20), although a few excep­
tional reports have described that the
anammox process can be operated steadily at
around 20℃ and at lower temperatures11),15),19)
as shown in Table 1. Dosta et al.6) reported
that low temperature cause irreversible
inhibition of anammox activity because of
NO2 －N accumulation.
As such an approach, adaptation of
temperatures is not always effective and
reproducible. Other trials have been un­
dertaken to start-up anammox reactors
directly under ambient or rather lower
temperatures21)－25) ( Table  2). Nevertheless,
such studies have been few. Especially,
anammox reactors with high nitrogen
removal performance under ambient tem­
perature have rarely been established. It is
therefore worthwhile to develop a novel
anammox reactor for use with wastewater
treatment at ambient temperature.
This study was conducted to start-up lab-

Table 1  Lab-scale anammox reactors operated under low temperature after enrichment under mesophilic conditions
Enrichment conditions Adapted conditions References
Reactor type Inoculum sources Temperature Temperature
SAA a or NRRb SAA a or NRRb
(℃) (℃)
Nitritation- Mature anammox 30 SAA 40 25 SAA 11.9－16.6 11)
anammox and aerobic
sequencing batch ammonium 12 SAA 11.4－18.4
reactor oxidizers
Up-flow Activated sludge 30 NRR 5.72 16 NRR 2.28 15)
anaerobic sludge
blanket
Up-flow Mature anammox 35 NRR 24.2 2.5－15.8 NRR 7.97 20)
anaerobic sludge sludge
blanket 10.7－21.6 NRR 4.52
a b 3
SAA, specific activity of anammox bacteria (nmol－N/mg－protein/min); NRR, nitrogen removal rate (kg－N/m /day).

Table 2  Lab-scale anammox reactors operated under ambient temperature

Time
a b
Temperature NLR NRR needed for
Reactor type Inoculum sources References
the NRR
(℃) (kg－N/m3 /day) (days)
Fixed-bed column reactor 25 0.08 0.06 500 Coastal sediment of Ago Bay, Japan 21)
with non-woven fabric
Up-flow fixed-bed column 20 3.3 2.17 550 Coastal sediment of Hiroshima Bay, 22)
with non-woven fabric sheet Japan
Up-flow column reactor 18－25 0.35 0.07－0.26 400 Freshwater sediments from rivers, 23)
packed with non-woven fabric lakes and dams, Japan
Gas-lift sequencing batch 10 － 0.027 700 Activated sludge mixture of waste 24)
reactor/Membrane bioreactor water treatment plants, Netherlands
Up-flow reactor packed with 15 2－3 1.55－2.18 800 Sludge of river, lake, dam reservoir 25)
nonwoven fabric material
Up-flow reactor packed with 20 0.9－1.0 0.64 800 Activated sludge of a domestic This study
nonwoven fabric material WWTP in Kumamoto, Japan
0.9－1.0 0.64 800 Activated sludge from a livestock
WWTP in Hokkaido, Japan
0.52 0.38 700 Microorganisms collected from
groundwater in Kumamoto City
a
NLR, nitrogen loading rate; bNRR, (maximum) nitrogen removal rate.
 Startup of Lab-scale Anammox Reactors Seeded with Activated Sludge at Ambient Temperature 75

scale anammox bioreactors at 20℃. Microbial
inocula for the bioreactors were two activated
sludge samples collected from wastewater
treatment plants in Kumamoto Prefecture in
the temperate region of Japan and in
Hokkaido Prefecture in the subarctic region
of Japan. In addition, a groundwater sample
was collected from the temperate region and
was used as the inoculum.
MATERIALS AND METHODS
Seed microorganisms    Seed sludge samples
for Reactors 1 and 2 were collected respec­
tively from a domestic wastewater treatment
plant ( WWTP) in Kumamoto City, and a
livestock WWTP in Hokkaido Prefecture.
Both of activated sludge samples were taken
from nitrification tanks. For Reactor 3,
microorganisms in several liters of ground­
water of Kumamoto City were collected by
filtration (1.0 µm pore size).
Synthetic wastewater    Synthetic inorganic
wastewater contained 54 mg KH2PO4 , 125 mg
NaHCO3 , 0.5  mL of Trace element solution I
( FeSO4 18  g/L, EDTA 10  g/L), and 1  mL of
Trace element solution II ( EDTA 15  µg/L,
ZnSO47H 2 O 0.430 µg/L, CuSO4•5H 2 O 0.250
µg/L, NiCl 2•6H 2 O 0.190 µg/L, NaMoO4•2H 2 O
0.22 µg/L, and H3BO4 0.014  µg/L) 26) in 1  L of
tap water. Synthetic wastewater for Reactors
1 and 2 also contained ( NH4) 2 SO4 20－125
mg－N/L and NaNO2 20－115  mg－N/L ( Table
3). Synthetic wastewater for Reactor 3
contained lower inorganic nitrogen as
( NH4) 2 SO4 10－60  mg－N/L and NaNO2 10－70
mg－N/L. The synthetic wastewater described
above was purged with N2 gas to achieve
dissolved oxygen ( DO) concentration lower
than 0.5  mg/L.
Reactor setup and operation    Three acrylic
column reactors (500  mL each) were used to
enrich anammox bacteria in this study as
depicted in Fig. 1. The reactors were packed
with a nonwoven fabric material of 0.7－cm
thickness porous polyester as the biomass
carrier (Japan Vilene Co. Ltd., Tokyo) which
occupied about 69% of the reactor volume to
enhance the retention of microorganisms.
Sludge samples were inoculated as 1000 mg－
MLSS to the biomass carrier to Reactors 1
and 2. The groundwater microorganisms on
the filter paper were inoculated to the
biomass carrier in Reactor 3.
Synthetic wastewater was fed continuously
by peristaltic pumps to the reactors in up-
flow mode. All reactors were kept at 20℃
under anaerobic conditions with hydraulic
retention time ( HRT ) of 6－17  h. The opera­
tional conditions of the reactors are presented
in Table  3. No data were recorded during
days 464－536 for Reactor 1, days 485－542 for
Reactor 2, or days 378－448 for Reactor 3
because of the absence of analytical staff
between academic years.
Analytical procedures    The NH4 －N, NO2
－N and nitrate ( NO3 －N ) concentrations were

Table 3  Influent inorganic nitrogen concentrations and

operational HRT
Period NH4 －N NO2 －N HRT
(days) (mg/L) (mg/L) (h)
Reactor 1 1－325 20 20 17
326－536 20－70 20－80 12.5
537－586 50 50 12
587－789 50－125 50－125 6
Reactor 2 1－325 20 20 17
326－575 20－70 20－80 12.5
576－696 50 50 12
697－815 50－115 50－115 6
Reactor 3 1－220 10 10 17
221－614 10－20 10－30 12.5
615－713 20－60 30－70 6 Fig. 1  Scheme of the 0.5 L anammox reactor.
76
measured using an ion-chromatography
system ( HIC-SP system; Shimadzu Corp.,
Kyoto). An AG4S-SC column ( Dionex Corp.,
CA) was used to measure the NO2 －N and
NO3 －N concentrations and the mobile phase
was a 3  mM Na 2CO3 solution. A column
(Shim-pack IC-C4; Shimadzu Corp., Kyoto)
was used for determination of the NH4 －N
concentration. The mobile phase was a 3.0
mM of (COOH ) 2 solution.
RESULTS
Nitrogen removal performance of Reactor 1   mg/L on days 587－789. Finally, Reactor 1
Fig. 2 presents time courses of the nitrogen
concentrations, the nitrogen loading rate
( NLR), the nitrogen removal rate ( NRR), and
the operational parameters of Reactor 1,
which was inoculated with activated sludge
obtained from a domestic WWTP in
Kumamoto City.
The nitrogen removal was not substantial
until day 200 in the reactor fed with
wastewater containing NH4 －N of 20  mg/L
and NO2 －N of 20 mg/L at HRT of 17 h. After
day 80, the NH4 －N concentration and the pH
value in effluent decreased, although the NO3
－N concentration increased, suggesting the
occurrence of nitrification. During days 220
－400, the NH4 －N removal decreased gradu­
ally. Both NO2 －N and NO3 －N were almost
completely removed. A considerable increase
in the NRR was observed. Subsequently,
HRT was shortened to 12.5  h on day 326.
The NLR increased from 0.077 to 0.29 kg－N/
m3 /day by increasing the influent NH4 －N and
NO2 －N concentrations during days 326－463.
After day 400, the NH4 －N removal increased
suddenly and the NO2 －N removal was stably
maintained. Consequently, the NRR also
increased from 0.09 to 0.25  kg－N/m3 /day
with increased effluent pH over 8.0,
suggesting the occurrence of substantial
anammox reaction. This inference was
supported by an increase in the effluent NO3
－N concentration during this period. The
nitrogen removal efficiency was 80%, on
average, during days 382－463. It reached
95% at the maximum against the NLR at
0.26 kg－N/m3 /day on day 446.
The reactor operation was maintained at
the same NLR but no data were recorded
during days 464－536. After a period with no
Japanese J. Wat. Treat. Biol. Vol.52 No.3
chemical analysis and precise operation, the
NH4 －N and NO2 －N concentrations in effluent
became high during days 537－586, but
decreased with shortened HRT from 12 h to
6 h on day 587. Although the inflow rate to
the reactors dropped temporarily on day 638
because of pump trouble and because the
reactor treatment performance was lowered,
the nitrogen removal recovered after the
difficulty was resolved. Subsequently, the
influent NH4 －N and NO2 －N concentrations
were adjusted gradually from 50 mg/L to 125
achieved NRR over 0.64 kg－N/m3 /day, when
the NLR was 1.0 kg－N/m3 /day on day 789.
Fig. 3 presents the appearance of Reactor 1.
The biomass on the nonwoven fabric material
turned to red at about day 400 and finally
turned to bright red on around day 800.
Nitrogen removal performance of Reactor 2  
Fig. 4 presents time courses of the nitrogen
concentrations, NLR, NRR, and the opera­
tional parameters of Reactor 2, which was
inoculated with activated sludge sampled
from a livestock WWTP in Hokkaido.
Reactor 2 showed nitrogen removal per­
formance that was similar to that of Reactor
1 throughout the operation. Until day 200
after the start-up, the decrease in the NH4 －N
and NO2 －N concentrations coupled with the
increase in the NO3 －N concentration in
effluent suggested the occurrence of nitrifi­
cation. The NRR began to increase after day
221. The NRR increased considerably when
HRT was shortened from 17 h to 12.5  h and
the NLR intensified to increase from 0.07 to
0.3 kg－N/m3 /day during days 329－575. The
NRR rose from 0.08 to 0.23  kg－N/m3 /day
from day 385, suggesting the occurrence of
the substantial anammox reaction. The
nitrogen removal in Reactor 2 was 73% on
average during days 385－484 and 90% at the
maximum on day 429 when the NLR was
0.21  kg－N/m3 /day. No data were obtained
during days 485－542. The influent NH4 －N
and NO2 －N concentrations were adjusted
gradually from 50 mg/L to 115 mg/L; HRT
was shortened from 12 h to 6 h during days
697－815. Finally, the NRR reached 0.64  kg－N/
m3 /day against the NLR at 0.92 kg－N/m3 /day
on day 815. The biomass on the nonwoven
fabric material in Reactor 2 also turned to
Startup of Lab-scale Anammox Reactors Seeded with Activated Sludge at Ambient Temperature 77

Fig. 2 Time courses of HRT, pH, nitrogen loading rate (NLR) and nitrogen removal rate
(NRR) of Reactor 1: (a) NH4－N – ● influent, ○ effluent; (b) NO2－N – ▲ influent,
△ effluent; (c) NO3－N – □ effluent; (d) ◆ NLR – ◇ NRR; (e) HRT; (f) pH – *
influent, + effluent.
78
Fig. 3 Strong reddish color of fabric carriers in Reactor
1 and Reactor 2: (a) Reactor 1 on day 477, (b)
Reactor 1 on day 850, (c) Reactor 2 on day
477, and (d) Reactor 2 on day 850.
bright red ( Fig. 3).
Nitrogen removal performance of Reactor 3
Fig. 5 shows the time courses of the nitrogen
concentrations, NLR, NRR, and the opera-
tional parameters of Reactor 3, which was
inoculated with groundwater microorganisms
sampled in Kumamoto City. Reactor 3 was
fed with synthetic inorganic wastewater
containing NH4 －N and NO2 －N of 10－70 mg/L
at HRT of 6－17 h for 713 days.
Although even 270 days had passed from
the start-up, the effluent NH4 －N concentration
still fluctuated at high values, whereas NO2
－N was removed stably during the same
period. The NRR remained very low, even
though it increased gradually to 0.01－0.03
kg－N/m3 /day. The NLR was set up to 0.06
kg－N/m3 /day on day 284. However, the NH4
－N removal was not significant. No data were
obtained during days 378－448. Although NH4
－N and NO2 －N were removed almost com-
pletely after day 450, the nitrogen removal
in Reactor 3 was unstable until day 614.
After HRT was adjusted to 6 h on day 615,
the NRR increased to 0.38 kg－N/m3 /day at
the NLR of 0.52 kg－N/m3 /day on day 713.
However, no marked color change of the
biomass was observed in Reactor 3. Finally,
the operation of Reactor 3 was stopped and
dismantled on day 714.
Nitrogen reaction ratio in the anammox reactors
The molar reaction ratio of almost pure cell
Japanese J. Wat. Treat. Biol. Vol.52 No.3
suspension of highly active anammox bacteria
is reported as NH4 －N consumption: NO2 －N
consumption: NO3 －N production=1: 1.22±0.08:
0.21±0.07 27). Against the NH4 －N consumption,
the NO2 －N consumption and the NO3 －N
production are shown for evaluation of the
reaction ratio in the reactors during the
period when substantial NH4 －N and nitrogen
removal was observed, as depicted in Fig. 6.
The ratios of NH4 －N consumption, the NO2
－N consumption, and the NO3 －N production
in Reactors 1 and 2 were almost identical
values of 1:1.21: 0.19 and 1:1.23: 0.19, respec-
tively. By contrast, the reaction ratio of
Reactor 3 was 1:1.30 : 0.48, which was only
slightly different from the typical anammox
value.
DISCUSSION
This study attempted to start-up the
anammox reactors at 20℃, aiming at
enrichment of anammox bacteria suitable for
nitrogen removal under ambient temperature.
Reactors 1 and 2 inoculated with activated
sludge samples from the WWTPs showed
significantly high NRRs after about 220 days
and simultaneous efficient removals of NH4
－N and NO2 －N after about 400 days. The
nitrogen reaction ratio in both reactors was
similar to the typical anammox value27). The
biomass showed bright red, which is specific
for anammox cells with a large amount of
cytochrome c 3), 28). Based on those results, the
anammox process was proved to be able to
establish at 20℃. Although Reactor 3
inoculated with groundwater microorganisms
also showed significant nitrogen removal
after 100 days, the NH4 －N removal was not
performed stably until 400 days. The
simultaneous removals of NH4 －N and NO2 －N
were achieved stably after 450 days,
suggesting the occurrence of anammox in
Reactor 3. However, the nitrogen reaction
ratio did not obey the typical anammox
formula. Biomass did not change to reddish
color during the 750 days experimental
period.
Therefore, the result probably cannot be
certain that anammox bacteria were enriched
also in Reactor 3, though relatively high
nitrogen removal by the anammox-like
reaction was eventually confirmed.
Startup of Lab-scale Anammox Reactors Seeded with Activated Sludge at Ambient Temperature 79

Fig. 4 Time courses in HRT, pH, NLR, and NRR of Reactor 2: (a) NH4－N – ● influent,
○ effluent; (b) NO2－N – ▲ influent, △ effluent; (c) NO3－N – □ effluent; (d) ◆
NLR – ◇ NRR; (e) HRT; (f) pH – * influent, + effluent.
80 Japanese J. Wat. Treat. Biol. Vol.52 No.3

Fig. 5 Time courses of HRT, pH, NLR, and NRR of Reactor 3: (a) NH4－N – ● influent, ○ effluent; (b) NO2－N – ▲
influent, △ effluent; (c) NO3－N – □ effluent; (d) ◆ NLR – ◇ NRR; (e) HRT; (f) pH – * influent, + effluent.
During days 400－450, the influent NH4－N concentration, influent NO2－N concentration, and HRT were
maintained, respectively, at 20 mg－N/L, 20 mg－N/L, and 12.5 h (Table 3).
 Startup of Lab-scale Anammox Reactors Seeded with Activated Sludge at Ambient Temperature
Fig. 6 Reaction ratio of the NH4－N consumption, NO2－N consumption and the NO3－N production in the
reactors: (a) Reactor 1, (b) Reactor 2, and (c) Reactor 3, respectively, after 382 days, 385 days,
and 620 days after start-up; open diamonds, NO2－N/NH4－N; open triangles, NO3－N/NH4－N.
These results demonstrated the ubiquitous
presence of anammox bacteria, which can be
enriched and which can exhibit their activity
at ambient temperatures such as 20℃.
However, the results obtained from this
study, it infers that activated sludge is a
better seed for the start-up of anammox
reactors under ambient temperature than
groundwater microorganisms: activated sludge
requires a shorter period to establish stable
and efficient anammox reaction.
The maximum NRR achieved in both
Reactors 1 and 2 was 0.64  kg－N/m3 /day.
Higher NRRs might be achieved, if higher
NLRs had been given to the reactors.
Although steady operation was not attained,
the maximum NRR of 0.38 kg－N/m3 /day was
observed in Reactor 3. However, the NRR at
0.98  kg－N/m3 /day at 30℃ was achieved in
the anammox bioreactor started up under the
mesophilic conditions with seed activated
sludge in Kumamoto like Reactor 129). The
NRR of Reactors 1 and 2 at 20℃ is sufficiently
high in comparison with the mesophilic
reactor because the temperature coefficient
(Q10) is generally 2 to 3 for most biological
reactions30). Consequently, the start-up of the
anammox reactor under ambient temperature
was confirmed to be a good strategy for
achieving efficient anammox treatment in
low-temperature conditions.
The NRR in the anammox reactors
achieved in this study is presented in Table 2
compared with that obtained in earlier
studies examining the enrichment of
anammox bacteria at low temperatures (10
－25℃). Actually Reactors 1 and 2 in this
81
study achieved much higher NRRs than
those attained in most other studies that
have assessed operations of anammox reactors
at low temperatures. For example, Osaka et
al.23) enriched anammox bacteria from river
sediments in a bioreactor at 18℃ for more
than 400 days, thereby achieving NRR
of only 0.07－0.14  kg－N/m3 /day. Furthermore,
Hendrickx et al.24) enriched anammox bacteria
in a lab-scale reactor at 10℃ using activated
sludge as the seed for more than 700 days,
finding NRR of merely 0.027  kg－N/m3 /day.
The NRR achieved in a bioreactor packed
with non-woven fabric that had been placed
at the sea floor in advance for enrichment
was 0.06 kg－N/m3 /day 21). Although Kamei et
al.25) reported exceptionally high NRRs at
15℃ (1.55－2.18  kg－N/m3 /day) in bioreactors
seeded with sludge collected from a river,
lake, and dam reservoir, those values were
recorded within only 5 days after 800 days
of enrichment: a non-steady state. Only
Kindaich et al.22) achieved a very high and
stable NRR over 2  kg－N/m3 /day at 20℃
within 550 days of enrichment in a synthetic
nutrient medium based on artificial seawater.
In their studies21), 22), marine sediments were
used as the seed for the bioreactor. Conse­
quently, the enriched anammox bacteria can
exhibit their activity only under high-salinity
conditions which are not ordinarily observed
in real wastewater treatment processes.
Results show that Reactors 1 and 2
established in this study offer the substantial
possibility of supporting efficient anammox
processes for nitrogen removal without
additional heating systems. The enriched
82
biomass in Reactors 1 and 2 will be studied
further to elucidate the temperature
sensitivity of the anammox activity and the
phylogenetic structure of the bacterial
community.
ACKNOWLEDGEMENT
This study was supported by NEDO
( New Energy and Industrial Technology
Development Organization), Japan.
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(Submitted 2016. 6. 22)
(Accepted 2016. 8. 31)