Bioresource Technology 335 (2021) 125225

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Fast start-up and enhancement of partial nitritation and anammox process

for treating synthetic wastewater in a sequencing bath biofilm reactor:
Strategy and function of nitric oxide
Ting Huang c, Jianqiang Zhao a, b, *, Sha Wang a, Lin Lei a
a
School of Water and Environment, Chang’an University, Xi’an 710064, Shaanxi, China
b
Key Laboratory of Subsurface Hydrology and Ecological Effect in Arid Region of Ministry of Education, Xi’an 710064, Shaanxi, China
c
School of Civil Engineering, Chang’an University, Xi’an 710064, Shaanxi, China

G R A P H I C A L A B S T R A C T

Biofilm formation AnAOB enhancement PN-A Stabilization PN-A Enhancement PN-A Enhancement
(day1-day10) Day(11-day30) (day31-day130) (day131-day160) (day161-day180)

AOB NOB AnAOB O2 O2 O2

O2 NH4
+
O2 NH4+ NO2- NO2- NO2-
NO2- NO2-
O2 NH4+ NH4+
Operation NO
NH4+

stage NO NH4 +
NH4+ NH4+ NH4+
NO NO NO
NO2- (16.0~45.0mgN·L-1) NO2- (0.6~20.0mgN·L-1) NO2- (1.2~3.0mgN·L-1) NO2- (1.1~6.0mgN·L-1) NO2- (1.5~3.0mgN·L-1)

Aeration mode Full aeration A/O/A O/A A/O/A O/A

Aeration rate 0.1L·min-1 0.1L·min-1 0.1L·min-1 0.2L·min-1 0.2L·min-1
Cycle time 12hours 12hours 12hours 8hours 6hours

Stage1: PN-A start-up Stage2: PN-A stabilization Stage3: PN-A Enhancement

0.40
0.10 10
TNLR, AnAOB activity (kg·m-3·d-1)

0.35 0
Dissolved NO（mg·L-1）

0.08 0.30 80
0.25
0.06
TNRE(%)

60
Performance 0.20

0.04 0.15 40
0.10
0.02 20
0.05

0.00 0.00 0
day1 day3 day10 day30 day31 day60 day120 day131 day160 day180
TNRR and TNRE represent total nitrogen loading rate and total nitrogen removal efficiency. A—Anaerobic, O—Oxic, A—Anoxic

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, the partial nitritation and anammox (PN-A) process was initiated within 30 days in a sequencing
PN-A batch biofilm reactor (SBBR) by employing pre non-aeration and post non-aeration with fixed aeration rates. The
Fast start-up average ammonia removal efficiency (ARE), total nitrogen removal efficiency (TNRE) of 98.5 ± 1.5% and 89.5 ±
Non-aeration
1.6% were achieved. By doubling aeration rate and agitation rate and adopting pre non-aeration, the TNRR was
Nitric oxide
Synergetic state
promoted from 0.135 ± 0.013 kg N⋅m− 3⋅d− 1 to 0.285 ± 0.015 kg N⋅m− 3⋅d− 1, obtaining an average ARE and
TNRE of 97.5 ± 1.5% and 85.5 ± 2.6%. Nitric oxide might induce anaerobic ammonia oxidation bacteria
(AnAOB) during the start-up stage, and could be an indicator for synergetic state between ammonia oxidation
bacteria (AOB) and AnAOB. Lower nitrous oxide emission factor of 0.51% was obtained. The abundance of AOB,
AnAOB and nitrite oxidation bacteria (NOB) accounted for 1.6%, 19.3% and 0.3%, respectively.

1. Introduction process, the PN-A process presents advantages including about 62.5%
less oxygen demand, 100% less external carbon source need, 90% less
Partial nitritation and anammox (PN-A) process, known as a prom­ surplus sludge production and zero carbon emission (Qiu et al., 2019).
ising alternative for nitrogen removal from wastewater, is under Presently, more than 80% of the existing full-scale anammox-based in­
development currently (Ali & Okabe, 2015; Lackner et al., 2014; Li et al., stallations are one-stage PN-A, because this configuration exhibits
2021; Perez et al., 2020). Compared to conventional nitrogen removal several conspicuous advantages including less land occupation, more

* Corresponding author at: School of water and environment, Chang’an University, Xi’an 710064, Shaanxi, China.
E-mail address: 626710287@qq.com (J. Zhao).

https://doi.org/10.1016/j.biortech.2021.125225
Received 10 March 2021; Received in revised form 22 April 2021; Accepted 23 April 2021
Available online 28 April 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
T. Huang et al.
simple operation and less substrate inhibition (Lackner et al., 2014).
One-stage PN-A has been widely implemented in treating high-
ammonium wastewater such as anaerobic digestion reject water,
where ammonium concentration was 400 ~ 5000 mg⋅L− 1 (Lackner
et al., 2014; Lackner & Horn, 2013; Szatkowska et al., 2007). The one-
stage PN-A process mainly includes two autotrophic pathways, where
ammonia oxidation bacteria (AOB) oxidize ammonium (NH+ 4 ) to nitrite
(NO–2) with oxygen, while anaerobic ammonia oxidation bacteria
(AnAOB) convert the remaining NH+ 4 and NO2 to dinitrogen gas (N2)
–
(Sliekers et al., 2003). However, low growth rates of AnAOB (doubling
time of 7 days to 30 days), different living conditions between AOB and
AnAOB, as well as NO–2 competition by nitrite oxidation bacteria (NOB)
cause difficulties for initiating a one-stage PN-A process, especially for
treating low-ammonium wastewater (Li et al., 2018; Lv et al., 2019).
Given low growth rates of AnAOB, biomass retention is critical for
implementing a one-stage PN-A process. Developing aggregate structure
such as biofilm has been proved to be an effective solution for AnAOB
biomass retention (Augusto et al., 2018; Cai et al., 2020; Li et al., 2021;
Yue et al., 2018). Besides, the layered structure of biofilm can provide
protection for AnAOB to resist unpleasant environmental factors such as
DO and substrate inhibition (Strous et al., 1999). The oxygen transfer
specificity of biofilm assures the simultaneity of nitritation and anam­
mox process, where NO–2 produced by AOB residing in the outer aerobic
layer could be immediately consumed by AnAOB residing in the inner
anoxic layer of the biofilm, assuring the co-culture of AOB and AnAOB
(Jiang et al., 2020; Li et al., 2021; Perez et al., 2020). However, even
abundant studies have been dedicated to starting up PN-A process, the
start-up duration and performance varied and the mechanism is still
unclear.
Additionally, NOB repression is critical for start-up and operation
stability of a PN-A process, especially for treating low-ammonium
wastewater (Li et al., 2018; Meng et al., 2019; Yue et al., 2018). To
solve this problem, the most crucial strategy was aeration control,
including limited DO control and intermittent aeration (Li et al., 2018).
Due to stronger DO affinity of AOB than NOB and longer lagging time of
NOB from non-aeration phase, intermittent aeration (DO during aera­
tion phase, 0.3 ~ 1.5 mg⋅L− 1) or limited DO (0.1 ~ 0.5 mg⋅L− 1) through
real-time DO control were usually employed for starting up and oper­
ating a PN-A process (Cai et al., 2020; Kowalski et al., 2018; Yue et al.,
2018). Whereas, NO–3 produced to NH+ 4 removed (△NO3-N/△NH4 -N)
– +
was usually more than stoichiometric value of 11.0% (Sliekers et al.,
2003) in those studies. Additionally, 50% of the surveyed plants expe­
rienced nitrate or nitrite build-up generally lasting up to several days
even under real-time DO control according to a survey report (Lackner
et al., 2014). Therefore, critical DO control could not assure the stability
of PN-A process in the long run. Zheng et al. enhanced PN-A process
elevating TNRR from 0.17 kg N⋅m− 3⋅d− 1 to 0.30 kg N⋅m− 3⋅d− 1 through
regulating aeration duration with the same aeration rate of 120 L⋅h− 1 in
an intermittent aeration system (Zheng et al., 2019). Li et al. achieved
long-term stability through maintaining NO–2-N concentration less than
1.0 mg⋅L− 1, where synergy between AOB and AnAOB was maintained
that NO–2 produced by AOB was simultaneously taken up by AnAOB
leaving low NO–2 in the liquid so as to provide possibility for NOB sup­
pression due to substrate limitation (Li et al., 2019). Hence, achieving
synergy between AOB and AnAOB is critical for the stability of PN-A
process. Therefore, more effective and reliable strategy rather than DO
concentration control strategy is on the way for starting up and imple­
menting a one-stage PN-A biofilm system.
In addition, it is confirmed that nitric oxide (NO) was a definite and
key intermediate of nitritation as well as anammox (Carantoa & Lan­
castera, 2018; Kuypers et al., 2018; Soler-Jofra et al., 2021). In nitrita­
tion process, according to recent studies, NH+ 4 is oxidized to
hydroxylamine by ammonia monooxygenase (Amo), hydroxylamine to
NO by hydroxylamine oxidoreductase (Hao) and NO to nitrite by
uncharacterized enzyme (Carantoa & Lancastera, 2018). In anammox
process, nitrite is firstly converted to NO by nitrite oxidase (Nir) enzyme
2
Bioresource Technology 335 (2021) 125225
and then hydrazine is synthesized from NO and NH+ 4 catalyzed by hy­
drazine synthase (HZS) (Kuypers et al., 2018). Besides, nitrous oxide
(N2O) is a certain product in the nitritation process and a well-known
potent greenhouse gas (265-fold higher than CO2) (Li et al., 2020;
Soler-Jofra et al., 2021). Moreover, it is reported that N2O emission in a
PN-A system is even higher than that in traditional nitrification/deni­
trification system (Li et al., 2020). Therefore, N2O emission is an
important evaluation index for PN-A process. However, although a large
number of studies have been devoted to the start-up and operation of
PN-A process, NO and N2O production was scarcely investigated.
Therefore, the purpose of current study was to realize fast start-up of
the PN-A process in an SBBR by regulating aeration duration through
employing pre non-aeration and post non-aeration with a fixed aeration
rate and higher initial AOB activity for treating low-ammonium waste­
water. The strategy leading to fast start-up, long term stable perfor­
mance and enhancement were discussed. Besides, dissolved NO and
dissolved N2O in typical cycles were monitored and analyzed. Further­
more, the abundance of functional bacteria including AOB, NOB and
AnAOB after start-up was evaluated by 16S rRNA high-through put gene
sequencing molecular biology. This study may provide a new sight for
fast start-up, enhancement, as well as N2O emission control of PN-A
process for treating ammonium-contained wastewater.
2. Materials and methods
2.1. Reactor description
A lab-scale SBBR reactor made of plexiglass with an effective volume
of 6 L and filled with combined packing (volume ratio of 5.0%) as bio­
film carriers was established. Two strings of ring-shaped biofilm carriers
of 10 cm diameters and combined packing with specific surface area of
1500 m2⋅m− 3 were fixed in the middle of each reactor. The reactor was
covered with opaque cloth to avoid light and the temperature was
controlled at 30 ± 2 ℃ with automatic thermostat heater (CJJ85-1,
AICE, China). Oxygen was supplied by air pressure pump (ACO-318,
HEILEA, China) with a disc diffuser. Aeration rate was controlled with a
rotameter. (LZB-3, YINHUAN, China). The hydraulic agitator (AT303S,
Atman, China) was used to increase the substrate transport capability.
The equipment for PN-A biofilm reactor is outlined in Fig. 1.
2.2. Synthetic wastewater and seed sludge
The basic synthetic wastewater contained 100 mgN⋅L− 1 of
NH4HCO3, 850 mg∙L− 1 of NaHCO3, 22.3 mg∙L− 1 of KH2PO4, 11.2
mg∙L− 1 of MgSO4⋅7H2O, 11.2 mg∙L− 1 of CaCl2 and 1 ml⋅L− 1 of trace
element stock solution. The trace elements were prepared and supplied
according to Wang et al. (Wang et al., 2019). The influent pH value was
maintained at 8.0 ± 0.5. The seed sludge was collected from aeration
tank and anoxic tank of a municipal wastewater treatment plants
(WWTPs) in Xi’an, China, mixed 1 to 1 by volume. Before set-up, the
inoculated sludge was aerated for 3 days to remove scum without any
substrate addition. The mixed liquor volatile suspended solids (MLVSS)
of the seeding sludge was 9.2 g VSS∙L− 1. The AnAOB activity of seed
sludge was 0.013 ± 0.023 kg TN⋅m− 3⋅d− 1.
2.3. Operation and experiment procedures
The experiment was implemented in three stages (Table 1). The
− 1
concentration of NH+ 4 -N was 100 mg⋅L . Aeration rate was determined
at 0.1 L⋅min− 1 (0.017 L air⋅L− 1⋅min− 1) with an initial ammonia oxida­
− 1 − 1
tion rate of 9.0 ± 1.5 mg NH+ 4 -N L ⋅h and DO concentration of 0.3 ±
0.2 mg⋅L− 1. The operation cycle contained feeding phase of 5 min,
reacting phase of 700 (460, 340) min, settling phase of 10 min and
draining phase of 5 min during different stages. Sodium acetate of 100
mg COD⋅L-1 was added in the influent in the first 10 days.
Water samples from influent and effluent were analyzed daily. The
T. Huang et al. Bioresource Technology 335 (2021) 125225

pH meter DO meter
Voltage acquisition host
N2O
NO 8.65mv
N2O 9.53mv

NO

Sampler
Heater controller
Inlet pipe
Heater
Water tank
Packing
(polymer fiber)
Outlet pump
Air meter

Hydraulic mixer

Submerge pump
Air pump disc diffuser

Fig. 1. Schematic diagram of the PN-A reactor.

Table 1
Operation strategies during each stages.
Stages Time Aeration mode Airflow rate (L⋅min− 1) (L Drainage TNLR Cycle time
(day) air⋅L− 1⋅min− 1)a ratio (kgN⋅m− 3⋅d− 1) (h)

stage1 1–10 Full aeration 0.1(0.017) 50% 0.100 12

(star-up stage) 11–30 Non-aeration(120 min)/aeration (480 min)/ non- 50% 0.100 12
aeration (100 min)
stage2 31–130 Aeration (600 min)/non-aeration (100 min) 0.1(0.017) 80% 0.160 12
(steady stage)
stage3 131–160 Non-aeration(60 min)/aeration (300 min)/non- 0.2(0.034) 80% 0.240 8
(enhancing aeration(100 min)
stage) 161–180 Aeration (300 min)/ non-aeration (40 min) 0.2(0.034) 80% 0.320 6
a
volume of air per volume of liquid per min.

pH of effluent and the average DO concentration during aeration phase gene. The amplicons were sent out to Sangon Co., Ltd., in Shanghai for
were monitored daily. AnAOB activities were examined on day 1, 30, 60, small-fragment library construction and pair-end sequencing using the
120, 160 and 180. In stage1, only the dissolved NO was monitored on illumine sequencing.
day 1, 3, 10, 30 in cycles. In stage2 (steady stage) and stage3 (enhancing
stage), nitrogen conversion including NH+ 4 -N, NO3-N, NO2-N, NO and
– –
2.6. Calculations
N2O, as well as oxidation reduction potential (ORP), pH and DO were
monitored synchronously at day 31, 60, 120, 131, 160 and 180. △NO–3-N/△NH+ 4 -N of each cycle was used to evaluate NOB build-up
extent. The △NO–3-N/△NH+ 4 -N, total nitrogen loading rate (TNLR),
total nitrogen removal rate (TNRR), total nitrogen removal efficiency
2.4. Analytical methods
(TNRE), ammonia removal rate (ARR), ammonia removal efficiency
(ARE), AnAOB activity and free ammonia (FA) were calculated ac­
NH+4 -N, NO3-N and NO2-N were measured daily according to the
– –
cording to Eqs. (1)-(8) (Li et al., 2017). Dissolved NO and dissolved N2O
standard methods (APHA, 2005). pH, DO and temperature were detec­
in liquid phase and the N2O emission were calculated according to Wang
ted with a portable meter (American Hach Company). Statistical cal­
et al. (Eqs. (9)-(10)) (Wang et al., 2019).
culations were performed using the SPSS 19.0 (IBM, USA) and OriginPro
9.0 (OriginLab, USA). Dissolved NO and N2O were detected using a (NO−3 − N)eff
Danish Unisense NO-500-111 807,724 microelectrode online moni­ ΔNO−3 − N/ΔNH4+ − N⋅(%) × 100 (1)
(NH4+ − N)inf − (NH4+ − N)eff
toring system. The AnAOB activities were examined by batch tests in suit
according to Zheng et al. (Zheng et al., 2019). ( ) TNinf × n × p
TNLR kg⋅N⋅m− 3 ⋅d− 1 = (2)
1000
2.5. Microbial community analysis ( ) (TNinf − TNeff ) × n × p
TNRR kg⋅N⋅m− 3 ⋅d− 1 = (3)
1000
Microbial community of the biofilm sample on day 180 was per­
formed through high throughput sequencing method, and the primers
515F/806R were selected for the V4 region of the 16S ribosomal RNA

3
T. Huang et al. Bioresource Technology 335 (2021) 125225

TNinf − TNeff ( ) ( )
TNRE (%) = × 100 (4) NH4+ − N inf − NH4+ − N eff
TNinf ARE (%) = ( ) × 100 (6)
NH4+ − N inf
(( ) ( ) )
( ) NH +
4 − N − NH +
4 − N ×n×p ( )
(5)
inf eff
ARR kg⋅N⋅m− 3 ⋅d− 1 = An AOB activity kg⋅TN⋅m− 3 ⋅d− 1 = (TNBI − TNBE ) × 24 × 10− 3
(7)
1000

Fig. 2. Start-up and enhancement of PN-A

process (TNLR represents total nitrogen
loading rate, ARR represents the ammonia
removal rate, TNRR represents total nitrogen
removal rate, ARE represents ammonia
removal efficiency, TNRE represents total
nitrogen removal efficiency, NAR represents
nitrite accumulation rate, △NO–3-N/△NH+ 4-
N represents nitrate produced to ammonia
removed. pH was detected in the effluent.
DO concentrations were the average value
during oxidation phase in cycles.)

4
T. Huang et al.
( )
( ) NH+
4 − N inf × 10
pH
FA mg⋅L− 1 = 6344 , t = 30◦ C (8)
e273+t + 10pH
re = kc (9)
∫t2
Q = Vw re dt (10)
t1
Where, NH+ 4 -Ninf and TNinf represent the influent ammonium nitrogen
and total nitrogen concentration, mg⋅L− 1, respectively. (NO–3-N)eff,
(NH+4 -N)eff and TNeff represent the nitrate nitrogen, ammonium nitrogen
and total nitrogen concentrations in the effluent respectively, mg⋅L− 1. n
and p present operation cycles per day and the drainage ratio. TNBI and
TNBE are total nitrogen concentrations in the influent and effluent of
AnAOB activity batch test, mg⋅L− 1. re is the N2O or NO emission rate,
mg⋅L− 1⋅h− 1, k is the N2O or NO emission coefficient, h− 1; c is the dis­
solved N2O or NO concentration, mg⋅L− 1; Q is the N2O or NO emission,
mg Vw is working volume of the reactor, L.
3. Results and discussion
3.1. Nitrogen removal performance
The nitrogen removal performance is shown in Fig. 2. During the first
10 days, full aeration and COD addition of 100 mg/L was applied.
Drainage ratio was 50% and the nitrogen loading rate (NLR) was 0.100
± 0.50 kg N⋅m− 3⋅d− 1. AOB activity was maintained high at the begin­
ning with ARE of 98.4%. DO concentration increased and ARE decreased
(Fig. 2), which is because oxygen utilization went down due to biofilm
resistance as biofilm developed. The biomass retention was 6.2 g
MLVSS⋅L− 1 at day 10, 62% of the initial 9.2 g MLVSS⋅L− 1. Meanwhile,
TNRE increased from 5.4% to 21.7%. NO–2-N in the effluent increased
first then decreased with an average nitrite accumulation rate (NAR) of
30.0%, while NO–3-N decreased from 73.1 mg⋅L− 1 to 27.6 mg⋅L− 1 grad­
ually. The results indicated that COD was consumed aerobically at the
beginning, while anammox or heterotrophic denitrification may occur
inside the biofilm afterwards and NOB was partially repressed.
Since day 11, non-aeration (120 min)/aeration (480 min)/ non-
aeration (100 min) mode was employed and COD addition was ceased
while other operation condition was unchanged. ARE decreased to
78.4% while the TNRE increased to 44.9% at day 11. The steep rise of
TNRE could be due to that pre-non-aeration and post non-aeration
provided conditions for AnAOB utilizing NO–2-N and NH+ 4 -N. Hetero­
trophic denitrification using autolysis of partial heterotrophic bacteria
due to sudden COD cut-off (Chen et al., 2016). During this stage, ARE
and TNRE gradually increased to 84.3% and 74.4%, respectively. The
NO–2-N concentration in the effluent was less than 1.0 mg⋅L− 1. The
△NO–3-N/△NH+ 4 -N decreased to 11.4% as NOB was inhibited gradu­
ally, which is close to stoichiometric value of 11.0% (Sliekers et al.,
2003). DO concentration decreased gradually and then stabilize at 1.5 ±
0.5 mg⋅L− 1, while effluent pH increased and then got stable at around
8.0. Biofilm changed from grey/brown to brick-red gradually, which
confirmed the occurrence of anammox morphologically. The effluent pH
increased gradually, as AnAOB enhanced and produced alkalinity, sug­
gesting the balance among AOB, NOB and AnAOB being gradually
established. The AnAOB activity was 0.113 ± 0.016 kgTN⋅m− 3⋅d− 1 at
day 30. The results suggested that AnAOB was successfully enhanced
and PN-A process was successfully realized within 30 days. The similar
start-up duration was achieved treating low-strength ammonia waste­
water in recent studies (Augusto et al., 2018; Cai et al., 2020; Yue et al.,
2018).
Since day 31, aeration mode was regulated to aeration (600 min)/
non-aeration (100 min) and the drainage ratio was raised to 80% with
the TNLR of 0.160 kg N⋅m− 3⋅d-1 to enhance the performance by pro­
moting aeration duration and NH+ 4 -N supply. ARE and TNRE declined by
5
Bioresource Technology 335 (2021) 125225
about 5.8% and 9.6% because of the change of NLR at the beginning and
gradually increased afterwards due to further AnAOB enhancement. DO
concentration exhibited a slightly decrease due to oxygen consumption
increasing. Since day 41, ARE and TNRE reached to 97.6% and 80.3%
and stabilized at 98.5 ± 1.5% and 89.5 ± 1.6% from day 60 to day 130.
△NO–3-N/△NH+ 4 -N was stabilized at 10.5 ± 0.6%, less than the stoi­
chiometric value of 11.0% (Sliekers et al., 2003), which might be
attributed to heterotrophic denitrification using extracellular polymeric
substance (EPS) or biodegradable organic compounds cell lysis (Augusto
et al., 2018). The AnAOB activities were 0.237 ± 0.021 kg N⋅m− 3⋅d− 1
and 0.245 ± 0.038 kg N⋅m− 3⋅d− 1at day 60 and day 120, respectively. pH
and DO concentrations kept stable during this phase. The results indi­
cated that the system showed an excellent stability of nitrogen removal
performance.
When the quality of effluent is stable and drainage ratio is un­
changed, it is necessary to reduce cycle time of SBBR to enhance ni­
trogen removal ability. However, just reducing cycle time will cause
substrate accumulated and result a low removal efficiency. According to
previous study, to enhance nitrogen removal ability of PN-A process
essentially, the precondition is to enrich AOB activity by increasing
oxygen, which would provide more nitrite for AnAOB, thereby
increasing nitrogen removal ability of PN-A (Li et al., 2018). Besides,
substrate transfer was very important for biofilm system and increasing
hydraulic agitator could increase nitrogen removal ability as reported
(Zheng et al., 2019). From day 131, the cycle time was reduced to 8 h
and the aeration rate was promoted to 0.2 L⋅min− 1 to enhance nitrogen
removal performance. The TNLR was increased from 0.160 kg
N⋅m− 3⋅d− 1 to 0.240 kg N⋅m− 3⋅d− 1 consequently. Additionally, hydraulic
agitator was doubled aiming at increasing substrate transfer. Aeration
mode was set to non-aeration (60 min)/aeration (300 min)/non-aera­
tion (100 min). As shown in Fig. 2, the DO concentration during aeration
phase was 2.0 ± 0.5 mg⋅L− 1 at day 131 and then declined and stabilized
at 1.6 ± 0.3 mg⋅L− 1 since day 139, which indicated AOB was enhanced
and consumed more oxygen. The effluent NH+ 4 -N increased to 11.3
mg⋅L− 1 at first and then deceased to less than 2.0 mg⋅L− 1 during this
period. The ARE and TNRE reached to 97.0 ± 1.5% and 85.0 ± 3.5%
since day 145 and the △NO–3-N/△NH+ 4 -N was 11.3 ± 0.5%. The ARR
and TNRR were 0.220 ± 0.015 kg N⋅m− 3⋅d− 1 and 0.203 ± 0.012 kg
N⋅m− 3⋅d− 1 after stabilization (day 145 to day 160) and the AnAOB ac­
tivity was 0.356 ± 0.043 kg N⋅m− 3⋅d− 1 at day 160. The results showed
that the PN-A process was successfully enhanced through promoting
aeration rate and intensifying agitation while NOB was successfully
suppressed by using anaerobic-oxic-anoxic aeration mode. In addition,
stable effluent pH and operation DO suggest that synergy among AOB,
NOB and AnAOB was maintained excellently.
From day 161, the cycle time was reduced to 6 h and the TNLR was
hence increased to 0.32 kg N⋅m− 3⋅d− 1. Aeration mode was set to aera­
tion (300 min)/ non-aeration (40 min). The DO concentration during
aeration phase exhibits a slightly decrease of about 0.2 mg⋅L− 1. The
△NO–3-N/△NH+ 4 -N increased to 14.0 ± 1.3% gradually. The effluent
NO–2-N was 1.2 ± 0.8 mg⋅L− 1 from day 165 to day 180. The AnAOB
activity increased to 0.363 ± 0.023 kg N⋅m− 3⋅d− 1 at day 180, obtaining
an average TNRE of 85.5 ± 2.6%. The results suggested that the PN-A
process was successfully enhanced.
3.2. Nitrogen conversion in typical cycles
3.2.1. Nitrogen conversion in cycles during steady stage
To investigate the synergy state of AOB, NOB and AnAOB after start-
up of the PN-A process, nitrogen conversion in typical cycles of the
experiment were analyzed (Fig. 3). At day 31 (Fig. 3(a)), the DO con­
centration was about 1.5 mg⋅L− 1, NO–2-N was 5.0 ± 1.2 mg⋅L− 1 during
− 1
aeration phase. NH+ 4 -N declined from 80.7 mg⋅L to 2.8 mg⋅L− 1, and
− 1 − 1
NO3-N increased from 1.3 mg⋅L
–
to 11.6 mg⋅L with △NO–3-N/
△NH+ 4 -N of 11.6%. At day 60 (Fig. 3(b)), the DO concentration kept
stable at 1.6 ± 0.8 mg⋅L− 1 while NO–2-N was 2.0 ± 0.3 mg⋅L− 1 in the first
T. Huang et al. Bioresource Technology 335 (2021) 125225

Fig. 3. Variations of NH+

4 -N, NO3-N and NO2-N, pH, DO, ORP, dissolved NO and dissolved N2O in typical cycles in steady stage on (a) day 31 (day1 of steady stage),
– –

(b) day 60 (day 30 of steady stage), (c) day 120 (day 90 of steady stage).

Fig. 4. Variations of NH+

4 -N, NO3-N and NO2-N, pH, DO, ORP, dissolved NO and dissolved N2O in typical cycles during enhancing stage (a) on day 131 (day 1 of
– –

enhancing stage), (b) day 160 (day 30 of enhancing stage), (c) day 180 (day 50 of enhancing stage).

6
T. Huang et al.
9 h before substrate was consumed up. Dissolved NO was 0.02 mg⋅L− 1 to
0.06 mg⋅L− 1 and the peak happened in the first 25 min of the typical
cycle, which is consistent with the conclusion of Anna et al. that NO
accumulation increased when ammonia concentration increasing in a
batch test (Ribera-Guardia & Pijuan, 2017). Dissolved N2O exhibited a
peak of 0.021 mg⋅L− 1 and then declined with the same trend with NO. At
day 120 (Fig. 3(c)), almost the same results were achieved as that at day
60. NO–2, NO, N2O, pH, DO and ORP nearly kept steady before substrate
was consumed up during the whole cycle, which indicated that a good
balance among AOB, NOB and AnAOB was kept. As can be seen from
Fig. 3(b)(c), the reactor experienced about 2-hour starvation and the PN-
A process exhibited excellent stability during day 60 to day 120.
3.2.2. Nitrogen conversion in cycles during enhancing stage
Nitrogen conversion in typical cycles were monitored during the
enhancing stage (Fig. 4). The DO concentration increased to 2.2 ± 0.2
mg⋅L− 1 due to the promoted aeration rate. The NO–2-N increased to 4.2 ±
1.2 mg⋅L− 1 during aeration period. NH+4 -N declined from 79.7 mg⋅L
− 1
− 1 − 1 − 1
8.6 mg⋅L , and NO3-N increased from 3.7 mg⋅L to 13.9 mg⋅L with
–
△NO–3-N/△NH+ 4 -N of 13.9% at day 131. Dissolved NO and ORP
exhibited a fluctuation, that average dissolved NO increased from 0.02
mg⋅L− 1 to 0.06 mg⋅L− 1 and the ORP showed an increasing trend during
the whole aerobic phase. The results might be because that the increased
oxygen inputs enriched AOB activity and the balance between AOB and
AnAOB was broken. Meanwhile, surplus NO–2-N and higher DO con­
centration may foster NOB. However, as experiment proceeding, AOB
and AnAOB activities were enhanced further, NO–2-N was decreased to
2.3 ± 0.5 mg⋅L− 1, NH+ 4 -N linearly decreased from 80.1 mg⋅L
− 1
to 1.2
− 1 − 1 − 1
mg⋅L , NO3-N increased from 1.7 mg⋅L to 11.9 mg⋅L with △NO–3-
–
− 1
N/△NH+ 4 -N of 10.8% and DO concentration was 1.8 ± 0.2 mg⋅L
day 160 (Fig. 4(b)), and the trend of NO, N2O and ORP tended to be
stable. At day 180 (Fig. 4(c)), NO–2-N was 2.7 ± 0.3 mg⋅L− 1, NH+ 4 -N
decreased from 81.1 mg⋅L− 1 to 0.45 mg⋅L− 1 and NO–3-N increased from
2.7 mg⋅L− 1 to 14.2 mg⋅L− 1 with △NO–3-N/△NH+ 4 -N of 14.1%. DO
concentration was 1.7 ± 0.3 mg⋅L− 1 and is slightly lower than that at day
160, which suggest that the oxygen consumption increased due to the
possibility of NOB recovery.
3.3. Microbial community
The microbial communities in the reactor at day 180 were studied
through high-throughput sequencing analysis. At phylum level (Fig. 5
(a)), the Planctomycetes (23.7%) was the dominant phylum in the
Fig. 5. Bacterial communities at the end of the experiment (day180). (a) at phylum level and (b) at genus level.
Bioresource Technology 335 (2021) 125225
biofilm, while the other phyla were Proteobacteria (17.7%), Bacter­
oidetes (9.4%), Acidobacteria (7.4%), Chloroflexi (6.2%), Ignavi­
bacteriaeand (3.4%), Armatimonadetes (16.3%), Verrucomicrobia
(1.7%) and Nitrospirae (0.3%). Planctomycetes, Proteobacteria and
Nitrospirae were mainly affiliated to AnAOB, AOB and NOB, respec­
tively. Bacteroidetes, Chloroflexi and Armatimonadetes were supposed
to play a significant role on anammox process (Guo & Zhang, 2012;
Speth et al., 2016), because Bacteroidetes and Chloroflexi contribute to
stable backbone for bacteria aggregates. According to previous studies,
Armatimonadetes can maintain the stable of biofilm aggregates avoiding
detachment by scavenging detritus and peptides produced by anammox
bacteria (Lawson et al., 2017).
At genus level (Fig. 5(b)), the most dominant nitrogen conversion
related genus was Candidatus Kuenenia (19.3%), while the other main
genera also included Nitrosomonas (1.6%), Nitrospirae (0.3%) and
Thauera (0.3%). Among these genera, Nitrosomonas, Candidatus Kuene­
nia and Nitrospirae affiliated to AOB, AnAOB and NOB were enriched in
to the PN-A biofilm process in consistent with previous study (Fan et al.,
2019; Kuypers et al., 2018; Yang et al., 2019). Candidatus Kuenenia was
the only AnAOB genus, which is because it showed a stronger substrate
affinity in one-stage PN-A process (Li et al., 2019; Zheng et al., 2019) in a
low nitrite accumulated system. As is well known that Thauera is a
denitrification genus, and it was the only heterotrophic denitrification
genus detected in this study, which may explain why △NO–3-N/△NH+ 4-
N was lower than the stoichiometric value of 11%, that denitrification
occurred using EPS or COD from cell lysis in this study. At genus level,
the main functional microbes including AOB and AnAOB affiliated
abundance accounted for 1.6%, and 19.3%, while the abundance of NOB
and heterotrophic denitrifier were 0.3% and 0.3% at day 180.
at Generally, microbial community analysis indicated that NOB was
suppressed effectively while AnAOB was successfully enriched and the
balance among functional microbes was established successfully. Stable
and solid backbone of biofilm was formed in this study, which is critical
for PN-A process while biofilm supplied a potent protection for AnAOB
from reverse condition as well as the precondition for synergy between
AOB and AnAOB. In brief, the results of microbial analysis are further
proof for stability of PN-A process in this study.
3.4. Strategy for fast start-up, stability and enhancement of PN-A process
Many strategies have been proposed for start-up of the PN-A process
(Augusto et al., 2018; Cai et al., 2020; Deng et al., 2016; Qiu et al., 2019;
Yue et al., 2018). Compared with those studies, the PN-A process was
7
T. Huang et al.
successful started up in a shorter period, gave a higher TNRR and
showed better stability in the long run in this study. This could be
attributed to the following aspects.
3.4.1. High biomass retention and functional bacteria enrichment
In this study, combined packing was used as biofilm carriers, which
has been proved to have an excellent adsorption capability with large
specific surface area (1500 m2⋅m− 3) (Deng et al., 2016; Li et al., 2018;
Yue et al., 2018). External COD of 100 mg⋅L− 1 (COD/N = 1) was added
to maintain enough biomass retention (Li et al., 2018). As expected,
almost all the seed sludge adhered to biofilm within 7 days except for
biomass loss from cell autolysis due to the condition change, resulting
biomass retention of 6.2 g MLVSS⋅L− 1 at day 10, 62% of the initial 9.2 g
MLVSS⋅L− 1. While in previous study, where no external COD was added,
the biomass retention on biofilm was only 45% of initial biomass and the
start-up time was more than 48 days (Cai et al., 2020). This was due to
that COD addition could facilitate biomass aggregation as well as
AnAOB enrichment (Li et al., 2021). Besides, different from gradual
enrichment of AOB in previous study by limiting DO concentration
(Augusto et al., 2018), higher initial ARE of 98.4% was obtained with
fixed aeration rate in the beginning. Moreover, enough gas agitation
could also promote microorganism aggregation and substrate transfer
(Jara-Muñoz et al., 2019; Jiang et al., 2020). Therefore, aeration rate of
0.1 L⋅min− 1 and COD addition of 100 mg⋅L− 1 might facilitate the fast
biofilm formation and efficient biomass retention, where AOB resided
outside and AnAOB resided inside the biofilm due to DO concentration
gradient provided by biofilm resistance, thus, contributing to fast start-
up of the PN-A process.
3.4.2. Synergy among AOB, NOB and AnAOB
Operation conditions including temperature, pH, and substrate are
also important for PN-A process (Meng et al., 2019). In this study, the
operation temperature and influent pH was maintained at 30 ± 2℃ and
8.0 ± 0.2, which have been proved to favor AOB outcompeting oxygen
with NOB as well as to benefit AnAOB (Li et al., 2018). In addition, the
FA concentration of the influent was 7.2 ± 0.3 mg⋅L− 1, which might be
another key factor for NOB suppression, since it has been reported that
AnAOB and AOB would be inhibited by FA of 54–178 mg⋅L− 1 and
10–150 mg⋅L− 1, while NOB would be inhibited by FA of 0.1–4.0 mg⋅L− 1
(Tomaszewski et al., 2017). However, even the most optimized, these
factors must be combined with aeration strategy (Augusto et al., 2018; Li
et al., 2018; Qiu et al., 2020).
Therefore, the key to start-up of PN-A process in the present study
was aeration strategy. A fixed aeration rate was employed, which
resulted a higher initial AOB activity, ensuring enough NO–2 supply to
AnAOB from the beginning, while in previous studies, DO concentra­
tions were maintained at a low level and low ARE was obtained at the
beginning (Augusto et al., 2018) resulting a low nitrogen removal per­
formance with TNRR of 0.08 kg N⋅m− 3⋅d− 1 and a longer start-up time of
46 days. From day 11 to day 30, pre-anaerobic phase prolonged the
sustainable time of relatively high FA concentration (5.6 ~ 7.5 mg⋅L− 1)
for 2 h, which reinforce the NOB suppression. In addition, pre-anaerobic
and post-anoxic phase provided condition for AnAOB enhancement,
since AnAOB could be enriched using NO–2-N accumulated in last cycle
or at the end of aeration phase, while drainage ratio of 50% was
employed. Consequently, synergy among AOB, NOB and AnAOB was
quickly established. Whereas, DO concentration control (0.1 ~ 0.5
mg⋅L− 1) was applied in previous reports (Augusto et al., 2018; Qiu et al.,
2020), where △NO–3-N/△NH+ 4 -N of 20%~40% was observed, longer
start-up time (more than 40 days) was needed, and lower TNRR of 0.024
~ 0.08 kg N⋅m− 3⋅d− 1 was obtained in the one-stage PN-A biofilm sys­
tems. However, the PN-A process was started up within 30 days and
TNRR of 0.095 kg N⋅m− 3⋅d− 1 was achieved here. Therefore, PN-A pro­
cess can be started up and synergy among AOB, NOB and AnAOB can be
established effectively by employing pre non-aeration and post non-
aeration with fixed aeration rate in a biofilm system, obtaining low
8
Bioresource Technology 335 (2021) 125225
NO–2 accumulation.
During stage 2, the system went through about 60-min aerobic
starvation and 100-min anaerobic starvation and low nitrite accumu­
lation was observed in each cycle while excellent synergy of functional
microbes and nitrogen removal was achieved. Ye et al. reported that
stable nitritation could be achieved by short term aerobic starvation (1
h) (Liu et al., 2017) and P. J. Reeve et al. concluded that AnAOB and
AOB could retained rapidly from a long period of starvation (62d)
(Reeve et al., 2016). Besides, AnAOB can recover from oxygen inhibition
rapidly (Strous et al., 1999). Therefore, stable synergy among AOB, NOB
and AnAOB could be partially attributed to aerobic starvation sup­
pressing NOB effectively in the long term. However, NOB robustness due
to imbalance between AOB and AnAOB would occur now and then in
over 50% of the surveyed PN-A system by conventional DO concentra­
tion control (Lackner et al., 2014). Matheus et al. achieved a △NO–3-N/
△NH+ 4 -N of 19.0% during the steady phase in a PN-A MBBR even under
low DO concentrations of less than 0.5 mg⋅L− 1 (Augusto et al., 2018).
Cai et al. attained △NO–3-N/△NH+ 4 -N of ＞15% using aeration (240
min)–non-aeration (240 min) mode in an SBBR system (Cai et al., 2020).
This is due to that DO could remain within the target control range when
oxygen input is higher than demand of AOB because of the consumption
by other microbes such as NOB (Qiu et al., 2020) or the synergy between
AOB and AnAOB was broken. Thereby, a gap between the oxygen de­
mand by AOB and input happened and NO–2 accumulation occurred. In
this study, the NOB suppression and stable performance were main­
tained sustainably over 90 days with unchanged aeration rate for an
average DO concentration of 1.5 mg⋅L− 1, achieving an average TNRE of
88.5%. Besides, the nitrogen variation in cycles was almost the same at
day 60 and day 120, which suggested that the synergy among AOB, NOB
and AnAOB was maintained well in the PN-A system with certain
aeration rate and stable influent, and the reason might be that anaerobic
and aerobic starvation could suppress NOB tellingly without AOB and
AnAOB recession. In sum, the synergy among AOB, NOB and AnAOB can
be established and maintained stable in this study, which cannot be
effectively guaranteed in DO concentration controlled system (Augusto
et al., 2018; Cai et al., 2020; Lackner et al., 2014; Yue et al., 2018).
Therefore, prolonging aerobic or aerobic starvation in an already syn­
ergetic PN-A system could maintain the stability of the PN-A system.
Notably, the precondition for applying aerobic starvation was that well
synergy was established between AOB and AnAOB.
3.4.3. Enhancement of PN-A process
In the present study, NOB was effectively suppressed before synergy
between AOB and AnAOB was reestablished by employing pre non-
aeration and post anoxic phase, when aeration rate was doubled. As a
result, TNLR of 0.220 ± 0.150 kg N⋅m− 3⋅d− 1 and an average TNRE of ＞
85.0% was obtained and got stable within 30 days. In previous study,
however, low DO concentration of＜0.5 mg⋅L− 1 was controlled and ni­
trogen substrate was doubled to promote nitrogen removal ability in a
MBBR PN-A system, nevertheless, NOB robustness occurred with
△NO–3-N/△NH+ 4 -N of more than 19.0% (Augusto et al., 2018). As re­
ported, NOB activity could recover in less than 30 min during aeration
phase in an aeration/non-aeration alternating system (Qiu et al., 2020).
Zheng et al. indicated that NOB could be successfully suppressed when
aeration time was 180 min with intermittent aeration mode (Zheng
et al., 2019). However, the aeration time in this study was more than
300 min. Therefore, other factors such as pre non-aeration might
contributed to NOB suppression significantly through prolonging dura­
tion of higher FA (about 7.0 mg⋅L− 1) and starvation before synergy
between AOB and AnAOB was established.
From day 161 to day 180, △NO–3-N/△NH+ 4 -N exhibited a slightly
increasing trend (from 11.0% to 14.5%) within 20 days, indicating that
NOB recovery occurred, which might be attributed to that pre non-
aeration was cancelled or aerobic (anaerobic) starvation was cut
down. This result further elucidated that pre non-aeration and duration
of anaerobic (aerobic) starvation was critical for NOB suppression.
T. Huang et al.
Besides, NOB could be suppressed but not be wiped out in this PN-A
system, because NOB would be recovered once conditions permitted
(Li et al., 2019).
3.5. NO and N2O analysis
3.5.1. The function of NO in a PN-A process
It was reported that NO was the product of NH2OH oxidation and
AOB denitrification in nitritation process (Carantoa & Lancastera,
2018). In recent studies, NO was supposed to be the virtual in­
termediates of AnAOB (Kuypers et al., 2018) and external NO can sup­
port AnAOB to grow together with NH+ 4 (Fig. 6) (Hu et al., 2019; Kartal
et al., 2010). Besides, Ivar Zekker et al. reported that NO (＜52 mg⋅L− 1)
could accelerate nitrogen removal rate and eliminate nitrite inhibition
to AnAOB (Zekker et al., 2015). In this study, the average NO accumu­
lation amounts increased in the first 10 days from 0.035 mg⋅L− 1 to
0.043 mg⋅L− 1 and then decreased from 0.025 mg⋅L− 1 at day 30. After
that, NO accumulation increased as drainage ratio was promoted at day
31, and declined to 0.015 mg⋅L− 1 as PN-A process established and ten­
ded stable from day 31 to day 120. This might be because NO produced
during the nitritation phase was used by gradually enriched AnAOB,
since AnAOB (Candidatus Kuenenia) could grow by utilizing external NO
to oxide NH+ 4 in the biofilm, and fluctuation of ammonium oxidation
rate could contribute to the variation of NO (Ribera-Guardia & Pijuan,
2017). Besides, it was reported that anammox could be completely
inhibited by nitrite at the concentration of 100 mgN⋅L− 1 in the anammox
system (Strous et al., 1999) while PN-A process could be initiated suc­
cessfully even with nitrite accumulation reaching to 100 ~ 500 mgN⋅L− 1
(van der Star et al., 2007). Moreover, it was summarized that it takes
longer time (45 day to 101 day) with initial NO–2-N of 50 mg⋅L− 1 and
− 1
NH+ 4 -N of 50 mg⋅L to start-up an anammox process even without ox­
ygen and substrate competition trouble (Chen et al., 2016; Deng et al.,
2016). Hence, it seems more difficult starting up an anammox process
than starting up a PN-A process when operation conditions are opti­
mized. This might be due to synergistic effect between AOB and AnAOB
in PN-A process, which might be virtually realized through NO produced
by AOB during the start-up stage. Moreover, aggregates structure of
biomass provide condition for interaction of AOB and AnAOB through
NO, since NO is instantaneously exist (Soler-Jofra et al., 2021), this may
essentially explain why PN-A process is realized more easily in the
granular and biofilm system.
Besides, when aeration rate was promoted suddenly at day 131, NO–2
increased and the average dissolved NO increased during the aerobic
phase from 0.015 mg⋅L− 1 at day 120 to 0.065 mg⋅L− 1 at day 131 (Fig. 4
(a)). This might be because the promoted aeration rates resulting a
NO, N2O
O2 Abiotic pathway O2 O2
AOB
Hao
Amo NH2OH NO Cu-Nir?
Nitritation
NH4+
Nir
Nxr
NO
N2 Hdh N2H4 Hzs NO3- Nor
AnAOB
Bioresource Technology 335 (2021) 125225
higher ammonia oxidation rate, causing imbalance between AOB and
AnAOB, that NO produced by AOB could not be synchronously
consumed by AnAOB. Afterwards, dissolved NO decreased and tend
stable as the balance between AOB and AnAOB was reestablished (Fig. 4
(b)(c)). It can be speculated that dissolved NO could be a nonnegligible
indicator for synergy between AOB and AnAOB. However, further and
more refinement study is needed to verify the point discussed above.
3.5.2. N2O accumulation analysis and reducing mechanism
N2O production in a PN-A process mainly occurs in three different
pathways, AOB denitrification, the hydroxylamine (NH2OH) oxidation
and heterotrophic denitrifiers denitrification (Fig. 6). AOB denitrifica­
tion pathway is mainly related to NO–2 concentration, while NH2OH
oxidation pathway is related to low DO concentration and fluctuation of
ammonia oxidation rate (Li et al., 2020; Soler-Jofra et al., 2021). As can
be seen from Fig. 3, dissolved N2O exhibited a peak in the first 25 min,
and then decreased during the following cycle time. The result is due to
that high ammonia oxidation rate at the beginning of oxic state after
transferring from anaerobic or anoxic state causes transient NH2OH
accumulation, increasing N2O production through NH2OH oxidation
pathway (Li et al., 2020; Soler-Jofra et al., 2021). As reported, N2O
production through AOB denitrification can be significantly enhanced
(N2O emission factor of 37.5%) during the nitritation process by NO–2
increase of 10 mg⋅L− 1 in a PN reactor (Tallec et al., 2006). However,
NO–2-N accumulated in a typical cycle during steady phase in this study
was only 2.8 ± 0.2 mg⋅L− 1, which interprets that dissolved N2O is
relatively low (about 0.02 mg⋅L− 1) after 25 min when NH2OH oxidation
reached balance in typical cycles. Besides, NO known as a precursor for
N2O in denitrification pathway, its instant consumption by AnAOB
might also contribute to reducing N2O production. As a result, the N2O
emission amount calculated according to Eq.9–10 is 0.044–0.051 mg⋅L-1
with an emission factor of 0.44 ~ 0.51%, which is much lower than that
reported in the PN-A process (0.98% to 4.5%) where low DO concen­
tration or intermittent aeration (30-min aeration time) strategy was
employed or higher NO–2-N was observed (Li et al., 2020; Ribera-Guardia
& Pijuan, 2017).
The results in this study indicated that one-stage PN-A process pro­
vides a prospect that N2O emission could be reduced markedly through
realizing synergy between AnAOB and AOB. To sum up, the strategy in
this study was outstanding in two aspects for reducing N2O production.
Firstly, long aeration duration (＞300 min) and higher DO (＞1.5
mg⋅L− 1) could weaken N2O production through NH2OH oxidation
pathway significantly, which cannot be realized in PN-A system using
limited DO concentration or intermittent aeration strategy (Li et al.,
2020). Secondly, synergy established between AOB and AnAOB make
Fig. 6. Main metabolic process and
O2 communication of key functional microor­
NOB ganisms involved in the PN-A process. The
Nxr
following enzymes perform the nitrogen
NO2- Nitrification
NO3- transformations: Amo (ammonia mono­
Nar oxygenase), Hao (hydroxylamine oxidore­
Nir Nir
ductase), Nor (nitric oxide reductases), Cu-
Nir? (an unknown enzyme that oxidize NO
to nitrite), Nxr (nitrite oxidoreductase), Nar
NO COD (nitrate reductase); Nir (nitrite reductase),
(Cell lysis and ESP) NOR (nitric oxide reductase), NosZ (nitrous
oxide reductase), Hzs (hydrazine synthase),
Nor Hdh (hydrazine dehydrogenase). Functional
HB bacteria: red dotted box, AnAOB (Candidatus
Kuenenia); blue dotted box, AOB (Nitro­
N2O NosZ N2 somonas); pink dotted box, NOB (Nitro­
spirae); Green dotted box, HB denitrififer
N2O (Thauera) (Kuypers et al., 2018). (For inter­
pretation of the references to colour in this
figure legend, the reader is referred to the
web version of this article.)
9
T. Huang et al.
sure that NO–2-N and NO produced by AOB might be consumed imme­
diately leaving relatively low NO–2-N and NO accumulation, which in­
terdicts condition for AOB denitrification pathway (Li et al., 2020).
Therefore, N2O production might be effectively controlled through
maintaining the balance and synergy of functional microbes in a one-
stage PN-A system.
4. Conclusions
In this study, the PN-A process was successfully started up and
enhanced in an SBBR with fixed aeration rates through establishing
synergy among AOB, NOB and AnAOB by employing pre non-aeration
and post anoxic periods and regulating drainage ratio properly. Low
NO–2 and NO accumulation due to synergy between AOB and AnAOB
contributed to maintaining stability of the PN-A process and reducing
N2O emission. NO accumulation could be an indicator for synergetic
state of AOB and AnAOB. Nitrosomonas and Candidatus Kuenenia affili­
ated to AOB and AnAOB genera were both enriched to an impressive
abundance of 1.6% and 19.3%.
CRediT authorship contribution statement
Ting Huang: Conceptualization, Project administration, Writing -
original draft, Writing - review & editing. Jianqiang Zhao: Supervision,
Funding acquisition, Writing - review & editing. Sha Wang: Investiga­
tion, Methodology. Lin Lei: Investigation, Methodology.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (no. 51778057) and the Key Research and Development Pro­
gram of Ningxia Hui Autonomous Region (no. 2019BFG02031).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2021.125225.
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