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[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
Graduate School of Engineering, Osaka University/2-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Department of Civil and Environmental Engineering, Faculty of Engineering, Kumamoto University
/2-39-1 Kurokami, Kumamoto 860-8555, Japan
Matsudo Research Center, Hitachi, Ltd., Infrastructure System Company
/537 Kami-hongo, Matsudo, Chiba 271-0064, Japan

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

requires an additional heating system to
Anaerobic ammonium oxidation (anammox) maintain 30-37℃ temperatures for efficient
is a biologically mediated reaction in which nitrogen removal5)-9), which lowers its energy
ammonium ( NH4 -N ) is oxidized to nitrogen efficiency.
gas using nitrite ( NO2 -N ) as the electron Consequently, the application of the
acceptor. Nitrogen removal processes using anammox process to wastewater treatment at
anammox1)-3) can provide important benefits ambient temperature (≤  25℃) has attracted
compared to conventional processes: less attention worldwide. Actually, anammox
oxygen demand, external carbon supply, and bacteria are reportedly present in the
sludge production. Recently, the anammox environment with diverse ranges of temper­
process has been implemented for treating ature: 10-43℃9). Furthermore, many reports
wastewater generated by some industries4). 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 temperatures is not always effective and
reports of the literature have described reproducible. Other trials have been un­
that lower temperatures of around 20℃ dertaken to start-up anammox reactors
severely damaged the specific anammox directly under ambient or rather lower
activity6),10),12),13),18)-20), although a few excep­ temperatures21)-25) ( Table  2). Nevertheless,
tional reports have described that the such studies have been few. Especially,
anammox process can be operated steadily at anammox reactors with high nitrogen
around 20℃ and at lower temperatures11),15),19) removal performance under ambient tem­
as shown in Table 1. Dosta et al.6) reported perature have rarely been established. It is
that low temperature cause irreversible therefore worthwhile to develop a novel
inhibition of anammox activity because of anammox reactor for use with wastewater
NO2 -N accumulation. treatment at ambient temperature.
As such an approach, adaptation of 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
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

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

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 Japanese J. Wat. Treat. Biol. Vol.52 No.3

measured using an ion-chromatography chemical analysis and precise operation, the

system ( HIC-SP system; Shimadzu Corp., NH4 -N and NO2 -N concentrations in effluent
Kyoto). An AG4S-SC column ( Dionex Corp., became high during days 537-586, but
CA) was used to measure the NO2 -N and decreased with shortened HRT from 12 h to
NO3 -N concentrations and the mobile phase 6 h on day 587. Although the inflow rate to
was a 3  mM Na 2CO3 solution. A column the reactors dropped temporarily on day 638
(Shim-pack IC-C4; Shimadzu Corp., Kyoto) because of pump trouble and because the
was used for determination of the NH4 -N reactor treatment performance was lowered,
concentration. The mobile phase was a 3.0 the nitrogen removal recovered after the
mM of (COOH ) 2 solution. difficulty was resolved. Subsequently, the
influent NH4 -N and NO2 -N concentrations
were adjusted gradually from 50 mg/L to 125
Nitrogen removal performance of Reactor 1   mg/L on days 587-789. Finally, Reactor 1
Fig. 2 presents time courses of the nitrogen achieved NRR over 0.64 kg-N/m3 /day, when
concentrations, the nitrogen loading rate the NLR was 1.0 kg-N/m3 /day on day 789.
( NLR), the nitrogen removal rate ( NRR), and Fig. 3 presents the appearance of Reactor 1.
the operational parameters of Reactor 1, The biomass on the nonwoven fabric material
which was inoculated with activated sludge turned to red at about day 400 and finally
obtained from a domestic WWTP in turned to bright red on around day 800.
Kumamoto City. Nitrogen removal performance of Reactor 2  
The nitrogen removal was not substantial Fig. 4 presents time courses of the nitrogen
until day 200 in the reactor fed with concentrations, NLR, NRR, and the opera­
wastewater containing NH4 -N of 20  mg/L tional parameters of Reactor 2, which was
and NO2 -N of 20 mg/L at HRT of 17 h. After inoculated with activated sludge sampled
day 80, the NH4 -N concentration and the pH from a livestock WWTP in Hokkaido.
value in effluent decreased, although the NO3 Reactor 2 showed nitrogen removal per­
-N concentration increased, suggesting the formance that was similar to that of Reactor
occurrence of nitrification. During days 220 1 throughout the operation. Until day 200
-400, the NH4 -N removal decreased gradu­ after the start-up, the decrease in the NH4 -N
ally. Both NO2 -N and NO3 -N were almost and NO2 -N concentrations coupled with the
completely removed. A considerable increase increase in the NO3 -N concentration in
in the NRR was observed. Subsequently, effluent suggested the occurrence of nitrifi­
HRT was shortened to 12.5  h on day 326. cation. The NRR began to increase after day
The NLR increased from 0.077 to 0.29 kg-N/ 221. The NRR increased considerably when
m3 /day by increasing the influent NH4 -N and HRT was shortened from 17 h to 12.5  h and
NO2 -N concentrations during days 326-463. the NLR intensified to increase from 0.07 to
After day 400, the NH4 -N removal increased 0.3 kg-N/m3 /day during days 329-575. The
suddenly and the NO2 -N removal was stably NRR rose from 0.08 to 0.23  kg-N/m3 /day
maintained. Consequently, the NRR also from day 385, suggesting the occurrence of
increased from 0.09 to 0.25  kg-N/m3 /day the substantial anammox reaction. The
with increased effluent pH over 8.0, nitrogen removal in Reactor 2 was 73% on
suggesting the occurrence of substantial average during days 385-484 and 90% at the
anammox reaction. This inference was maximum on day 429 when the NLR was
supported by an increase in the effluent NO3 0.21  kg-N/m3 /day. No data were obtained
-N concentration during this period. The during days 485-542. The influent NH4 -N
nitrogen removal efficiency was 80%, on and NO2 -N concentrations were adjusted
average, during days 382-463. It reached gradually from 50 mg/L to 115 mg/L; HRT
95% at the maximum against the NLR at was shortened from 12 h to 6 h during days
0.26 kg-N/m3 /day on day 446. 697-815. Finally, the NRR reached 0.64  kg-N/
The reactor operation was maintained at m3 /day against the NLR at 0.92 kg-N/m3 /day
the same NLR but no data were recorded on day 815. The biomass on the nonwoven
during days 464-536. After a period with no 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 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
Fig. 3 Strong reddish color of fabric carriers in Reactor
1 and Reactor 2: (a) Reactor 1 on day 477, (b) slightly different from the typical anammox
Reactor 1 on day 850, (c) Reactor 2 on day value.
477, and (d) Reactor 2 on day 850.
This study attempted to start-up the
bright red ( Fig. 3). anammox reactors at 20℃, aiming at
Nitrogen removal performance of Reactor 3 enrichment of anammox bacteria suitable for
Fig. 5 shows the time courses of the nitrogen nitrogen removal under ambient temperature.
concentrations, NLR, NRR, and the opera- Reactors 1 and 2 inoculated with activated
tional parameters of Reactor 3, which was sludge samples from the WWTPs showed
inoculated with groundwater microorganisms significantly high NRRs after about 220 days
sampled in Kumamoto City. Reactor 3 was and simultaneous efficient removals of NH4
fed with synthetic inorganic wastewater -N and NO2 -N after about 400 days. The
containing NH4 -N and NO2 -N of 10-70 mg/L nitrogen reaction ratio in both reactors was
at HRT of 6-17 h for 713 days. similar to the typical anammox value27). The
Although even 270 days had passed from biomass showed bright red, which is specific
the start-up, the effluent NH4 -N concentration for anammox cells with a large amount of
still fluctuated at high values, whereas NO2 cytochrome c 3), 28). Based on those results, the
-N was removed stably during the same anammox process was proved to be able to
period. The NRR remained very low, even establish at 20℃. Although Reactor 3
though it increased gradually to 0.01-0.03 inoculated with groundwater microorganisms
kg-N/m3 /day. The NLR was set up to 0.06 also showed significant nitrogen removal
kg-N/m3 /day on day 284. However, the NH4 after 100 days, the NH4 -N removal was not
-N removal was not significant. No data were performed stably until 400 days. The
obtained during days 378-448. Although NH4 simultaneous removals of NH4 -N and NO2 -N
-N and NO2 -N were removed almost com- were achieved stably after 450 days,
pletely after day 450, the nitrogen removal suggesting the occurrence of anammox in
in Reactor 3 was unstable until day 614. Reactor 3. However, the nitrogen reaction
After HRT was adjusted to 6 h on day 615, ratio did not obey the typical anammox
the NRR increased to 0.38 kg-N/m3 /day at formula. Biomass did not change to reddish
the NLR of 0.52 kg-N/m3 /day on day 713. color during the 750 days experimental
However, no marked color change of the period.
biomass was observed in Reactor 3. Finally, Therefore, the result probably cannot be
the operation of Reactor 3 was stopped and certain that anammox bacteria were enriched
dismantled on day 714. also in Reactor 3, though relatively high
Nitrogen reaction ratio in the anammox reactors nitrogen removal by the anammox-like
The molar reaction ratio of almost pure cell 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 81

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 study achieved much higher NRRs than
presence of anammox bacteria, which can be those attained in most other studies that
enriched and which can exhibit their activity have assessed operations of anammox reactors
at ambient temperatures such as 20℃. at low temperatures. For example, Osaka et
However, the results obtained from this al.23) enriched anammox bacteria from river
study, it infers that activated sludge is a sediments in a bioreactor at 18℃ for more
better seed for the start-up of anammox than 400 days, thereby achieving NRR
reactors under ambient temperature than of only 0.07-0.14  kg-N/m3 /day. Furthermore,
groundwater microorganisms: activated sludge Hendrickx et al.24) enriched anammox bacteria
requires a shorter period to establish stable in a lab-scale reactor at 10℃ using activated
and efficient anammox reaction. sludge as the seed for more than 700 days,
The maximum NRR achieved in both finding NRR of merely 0.027  kg-N/m3 /day.
Reactors 1 and 2 was 0.64  kg-N/m3 /day. The NRR achieved in a bioreactor packed
Higher NRRs might be achieved, if higher with non-woven fabric that had been placed
NLRs had been given to the reactors. at the sea floor in advance for enrichment
Although steady operation was not attained, was 0.06 kg-N/m3 /day 21). Although Kamei et
the maximum NRR of 0.38 kg-N/m3 /day was al.25) reported exceptionally high NRRs at
observed in Reactor 3. However, the NRR at 15℃ (1.55-2.18  kg-N/m3 /day) in bioreactors
0.98  kg-N/m3 /day at 30℃ was achieved in seeded with sludge collected from a river,
the anammox bioreactor started up under the lake, and dam reservoir, those values were
mesophilic conditions with seed activated recorded within only 5 days after 800 days
sludge in Kumamoto like Reactor 129). The of enrichment: a non-steady state. Only
NRR of Reactors 1 and 2 at 20℃ is sufficiently Kindaich et al.22) achieved a very high and
high in comparison with the mesophilic stable NRR over 2  kg-N/m3 /day at 20℃
reactor because the temperature coefficient within 550 days of enrichment in a synthetic
(Q10) is generally 2 to 3 for most biological nutrient medium based on artificial seawater.
reactions30). Consequently, the start-up of the In their studies21), 22), marine sediments were
anammox reactor under ambient temperature used as the seed for the bioreactor. Conse­
was confirmed to be a good strategy for quently, the enriched anammox bacteria can
achieving efficient anammox treatment in exhibit their activity only under high-salinity
low-temperature conditions. conditions which are not ordinarily observed
The NRR in the anammox reactors in real wastewater treatment processes.
achieved in this study is presented in Table 2 Results show that Reactors 1 and 2
compared with that obtained in earlier established in this study offer the substantial
studies examining the enrichment of possibility of supporting efficient anammox
anammox bacteria at low temperatures (10 processes for nitrogen removal without
-25℃). Actually Reactors 1 and 2 in this additional heating systems. The enriched
82 Japanese J. Wat. Treat. Biol. Vol.52 No.3

biomass in Reactors 1 and 2 will be studied anammox bacterium from a rotating

further to elucidate the temperature biological contactor treating ammonium-
sensitivity of the anammox activity and the rich leachate. Arch. Microbiol., 175, 198
phylogenetic structure of the bacterial -207 (2001)
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ACKNOWLEDGEMENT Ammonium removal from concentrated
This study was supported by NEDO waste streams with the anaerobic
( New Energy and Industrial Technology ammonium oxidation (Anammox) process
Development Organization), Japan. in different reactor configurations. Water
Res., 31, 1955-1962 (1997)
 9 ) Strous, M., Kuenen, J. G., and Jetten, M. S.
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