Bioresource Technology 101 (2010) 8074–8082

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

Nitritation and denitritation of domestic wastewater using a continuous

anaerobic–anoxic–aerobic (A2O) process at ambient temperatures
Wei Zeng, Lei Li, Yingying Yang, Shuying Wang, Yongzhen Peng *
Key Laboratory of Beijing for Water Environment Recovery, Department of Environmental Engineering, Beijing University of Technology, 100124 Beijing, China

a r t i c l e i n f o a b s t r a c t

Article history: In a continuous anaerobic–anoxic–aerobic (A2O) process treating domestic wastewater at ambient tem-
Received 10 March 2010 peratures, nitritation was achieved through a combination of short aerobic actual hydraulic retention
Received in revised form 14 May 2010 time (AHRT) and low dissolved oxygen (DO) levels (0.3–0.5 mg/L). The nitrite accumulation rate was
Accepted 29 May 2010
about 90% and ammonia removal efficiency was over 95%. With respect to total nitrogen removal, nitri-
Available online 25 June 2010
tation–denitritation at low DO levels of 0.3–0.5 mg/L was essentially equal to the complete nitrification–
denitrification at DO levels of 1.5–2.5 mg/L with the addition of external carbon sources. Regardless of
Keywords:
low DO operation, sludge bulking did not occur since the sludge volume index was below 150 ml/g.
Anaerobic–anoxic–aerobic (A2O) process
Nitritation
Real-time PCR assays showed that in response to complete and partial nitrification modes, the numbers
Ammonia oxidizing bacteria (AOB) of ammonia oxidizing bacteria population were 5.28  109 cells/g MLVSS and 3.95  1010 cells/g MLVSS,
Domestic wastewater respectively. Achievement of nitritation–denitritation is highly beneficial to the treatment of domestic
Real-time PCR wastewater in terms of lower carbon requirements and reduced aeration costs.
Ó 2010 Published by Elsevier Ltd.

1. Introduction
The conventional biological nitrogen removal is accomplished
by a two-stage treatment, i.e. the oxidation of ammonia to nitrite,
and then to nitrate in the first aerobic nitrification stage followed
by the second anoxic denitrification stage related to the reduction
of nitrate to nitrite, and then to N2 gas (Zhu et al., 2008). Nitrite is
an intermediate in both nitrification and denitrification. Therefore,
nitrogen removal via nitrite as an innovative technology was inves-
tigated and reported to be technically feasible and economically
favorable. The partial nitrification of ammonia to nitrite has been
named nitritation, and the subsequent direct reduction of nitrite
to N2 gas, denitritation (Jenicek et al., 2004). The application of nit-
ritation–denitritation could lead to a considerable saving in aera-
tion costs and external carbon sources as compared to the
complete nitrification–denitrification (Zhu et al., 2008; Fux et al.,
2006). For the treatment of domestic wastewater, nitritation and
denitritation are particularly significant because the organic car-
bon source in it is typically limiting (Zeng et al., 2003).
The key to achieve nitritation–denitritation is the control of oxi-
dation of ammonia to nitrite instead of nitrate and the build-up of
nitrite. Therefore, nitrite oxidizing bacteria (NOB) have to be inhib-
ited or eliminated while ammonia oxidizing bacteria (AOB) have to
become the dominant nitrifying bacteria (Hellinga et al., 1998). The
factors affecting nitrite build-up and the community structure such
* Corresponding author. Tel./fax: +86 10 67392627.
E-mail addresses: zengwei_1@263.net (W. Zeng), pyz@bjut.edu.cn (Y. Peng).
as higher temperature (Hellinga et al., 1998; Yoo et al., 1999), high-
er free ammonia (FA) and free nitrous acid (FNA) concentration
(Vadivelu et al., 2007; Peng et al., 2008; Park and Bae, 2009), inhib-
itor (Grunditz et al., 1998), low dissolved oxygen (DO) concentra-
tion (Ruiz et al., 2006; Blackburne et al., 2008; Guo et al., 2009),
control of hydraulic retention time (HRT) or aeration time (Peng
et al., 2007; Gao et al., 2009; Zeng et al., 2009) have been investi-
gated. Natural temperatures of domestic wastewater vary in the
range of 10–25 °C with the seasons. In this temperature range, high
nitrite build-up is difficult to maintain because the specific growth
rate of NOB is higher than that of AOB (Hellinga et al., 1998). The
FA and FNA concentrations in domestic wastewater cannot reach
levels that are inhibitory to NOB. Consequently, control of both
DO concentration and HRT seems to be the only available method
to establish nitritation. AOB have a stronger affinity to oxygen than
NOB at low DO concentrations, which causes nitrite build-up as
well as a savings in aeration cost (Garrido et al., 1997; Pollice
et al., 2002).
Low DO has been recognized as one of the key factors in simul-
taneous nitrification and denitrification (SND) (Zeng et al., 2003).
SND means that nitrification and denitrification occur concurrently
in one reactor under aerobic conditions. Both low DO levels and
nitritation promote the occurrence of SND via nitrite, which would
further reduce carbon requirements, aeration costs and waste
sludge (Ruiz et al., 2003; Zeng et al., 2003). Previous research also
showed that the extension of aeration time encouraged the transi-
tion of partial nitrification to complete nitrification and resulted in
the loss of nitrite build-up (Gao et al., 2009; Zeng et al., 2009). It

0960-8524/$ - see front matter Ó 2010 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2010.05.098
 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082
has been demonstrated that the control of HRT or aeration time is
an effective strategy for achieving nitrogen removal via nitrite
(Peng et al., 2007; Lemaire et al., 2008; Gao et al., 2009; Zeng
et al., 2009).
Process type also affects nitrite build-up. Two types of processes
are usually applied for biological nitrogen removal from wastewa-
ter, i.e. sequencing batch reactor (SBR) and continuous-flow sys-
tem. Yoo et al. (1999) suggested that alternating aerobic–anoxic
SBR could favor nitrite build-up. Presently, most of the studies on
nitritation and denitritation focus on the SBR process (Ruiz et al.,
2003; Zeng et al., 2003; Fux et al., 2006; Peng et al., 2007; Gao
et al., 2009; Zeng et al., 2009). In comparison with SBR, it is difficult
to achieve nitrite accumulation with the continuous. Only a few
studies on achieving nitrogen removal via nitrite in a continuous
process for treatment of wastewater with high ammonia concen-
trations (Ruiz et al., 2003; Peng et al., 2008) and anoxic–aerobic
(A/O) processes (Ma et al., 2009) have been carried out. Therefore,
nitritation and denitritation of domestic wastewater at ambient
temperatures using a continuous anaerobic–anoxic–aerobic (A2O)
process, which is most commonly used in wastewater treatment
plants (WWTPs) for biological nutrient removal, should be investi-
gated further.
Community structure, abundance and activities of AOB play an
important role in biological nitrogen removal from wastewater.
Presently, real-time polymerase chain reaction (PCR), as a rapid
and reliable quantitative method, has been applied to monitor
AOB for more efficient nutrient removal (Dionisi et al., 2003;
Harms et al., 2003; Araki et al., 2004; Limpiyakorn et al., 2005). A
fluorescence labeled probe (Okano et al., 2004) or SYBR Green (a
fluorescent dye for the quantification of double stranded DNA)
(Araki et al., 2004) is used for real-time quantitative PCR through
measuring fluorescence emitted continuously during the amplifi-
cation reaction. An external standard curve is the pre-requirement
of quantification, which shows the relationship between DNA copy
numbers and threshold cycle (Ct) values. Previous studies showed
that the standard curve was made using genomic DNA extracted
from pure culture of target bacteria (Okano et al., 2004), the plas-
mid carrying target gene (Harms et al., 2003) or synthesized DNA
fragment (Dionisi et al., 2002).
Therefore, this study aims to (1) investigate the effects of DO
concentration and HRT on nitrogen removal in an A2O process,
and achieve nitritation–denitritation of domestic wastewater at
ambient temperatures, (2) determine the mechanism of nitrita-
tion–denitritation, particularly with regard to the effect of low
DO concentration and short aerobic actual HRT, and (3) develop a
real-time polymerase chain reaction (PCR) assays for AOB quantifi-
cation to correlate AOB numbers with nitrogen removal.
Fig. 1. The schematic of the A2O process (1. influent tank; 2. pump; 3. DO and pH meter; 4. mixer; 5. pre-anoxic zone; 6. anaerobic zone; 7. anoxic zone; 8. aerobic zone; 9.
settler; 10. effluent; 11. air pump; 12. airflow meter; 13. air diffuser; 14. internal recycle; 15. external recycle; 16. waste sludge).
8075
2. Methods
2.1. Experimental set-up and operation
Fig. 1 shows the experimental system consisting of an A2O reac-
tor with a working volume of 80 L and a secondary settler with a
working volume of 24 L. The A2O reactor was divided into six
chambers. The first four chambers with mechanical mixers were
used as anaerobic or anoxic zones and the last two with air diffus-
ers were used as aerobic zones. The first chamber was a pre-anoxic
zone for denitrification of returned sludge (external recycle) from
secondary settler. The second chamber provided an anaerobic zone
for phosphorus release and for influent. The third and fourth cham-
bers were anoxic zones for denitrification of nitrite/nitrate recircu-
lation (internal recycle) from the last aerobic chamber. The volume
ratio of the pre-anoxic zone to anaerobic to anoxic to aerobic was
1:2:3:6. The flow rates of feed, returned sludge and nitrate recircu-
lation were controlled by peristaltic pumps. Each aerobic chamber
was equipped with one DO probe and one pH sensor. The airflow
meter controlled the aeration rate to achieve the desired DO con-
centration according to the different experimental conditions.
Temperature in the reactor was maintained at 22 ± 1 °C using a
heater and thermostat. The sludge retention time (SRT) was con-
trolled at 15–20 d by discharging an appropriate amount of settled
sludge. The mixed liquor suspended solid (MLSS) concentration
was about (2500 ± 500) mg/L.
2.2. Wastewater and sludge
Raw wastewater from a campus sewer line was pumped into a
storing tank for sedimentation, and then fed into the reactor. The
influent characteristics are shown in Table 1. Mean influent COD
to nitrogen ratio (C/N) was only about 2.5, and thus the organic
carbon source was typically limiting.
The seed sludge was taken from the Gao bei dian wastewater
treatment plant in Beijing, which utilizes a typical anoxic–aerobic
process to treat municipal wastewater and performs nitrification–
denitrification well. The wastewater composition and process is
similar to that used in this study. After sludge acclimation for
one month, a stable performance was achieved and the experi-
ments were begun.
2.3. Experimental procedure
The A2O system was operated for 180 d including six successive
phases (Table 2). Phase 1 (1–16 d) was to show the performance of
complete nitrification–denitrification at a DO concentration of
8076 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082

Table 1 nal recycle ratio, respectively; qðNO 

2 -NÞ is NO2 -N concentration in
Characteristics of the raw wastewater. effluent of the last aerobic zone; qðNO -NÞ is the sum of NO
x 2 -N and
Contents Range Average NO3 -N in effluent of the last aerobic zone. FA concentration was cal-
COD (mg/L) 105.4–270.6 177.5
culated as formula (4) (Anthonisen et al., 1976):
NHþ4 -N ðmg=LÞ
55.5–80.7 69.4
TN (mg/L) 59.6–85.6 73.7
½NHþ4 -N  107:6
FA ðmg=LÞ ¼ ð4Þ
C/N 1.84–3.85 2.53 exp½6334=ð273 þ TÞ þ 107:6
pH 7.12–7.46 7.29

2.5. Real-time PCR assay for quantification of AOB

1.5–2.5 mg/L and without the addition of external carbon sources.
Based on the operation in phase 1, sodium acetate (NaAc) and so-
dium propionate (COD of 150 mg/L) were supplied as the carbon
sources for denitrification in phase 2 (17–36 d). Both phases were
used for comparison with phases 4–5 that performed nitritation–
denitritation. The experimental purpose of phase 3 (37–76 d) was
to investigate if nitritation could be achieved by control of low
DO concentrations only. Phase 4 (77–124 d) was to investigate
the impact of a combination of low DO levels and HRT control on
the establishment of nitritation. Phase 5 (125–164 d) was to set
up a control strategy to further improve TN and ammonia removal
based on a stable performance of nitritation–denitritation. To find
the mechanism of partial nitrification, the aerobic actual hydraulic
retention time (AHRT) was extended in phase 6 (165–180 d) to
demonstrate the effect of aerobic AHRT control on the stable per-
formance of nitritation. NaAc (COD of 50 mg/L) was added as car-
bon sources for denitrification in phase 6.
2.4. Analytical methods
Chemical oxygen demand (COD), ammonia (NHþ 4 -N), nitrate
(NO 
3 -N), nitrite (NO2 -N), total nitrogen (TN, Jena Multi N/C
3000, Germany), sludge volume index (SVI), MLSS and mixed
liquor volatile suspended solid (MLVSS) were measured according
to the APHA Standard methods (1998). DO and pH were measured
on-line using DO/pH meters (WTW Multi 340i, Germany). An
Olympus BX52 microscope was used to observe the sludge flocs.
The aerobic AHRT, anoxic AHRT and nitrite accumulation
rate were calculated according to the following formulas
(Tchobanoglous et al., 2003):
Vo
aerobic AHRT ¼
Q ð1 þ R þ r Þ
VA
anoxic AHRT ¼
Q ð1 þ R þ r Þ
qðNO2 -NÞ
nitrite accumulation rate ¼
qðNOx -NÞ
where Vo and VA is the volume of aerobic and anoxic zone, respec-
tively; Q is the flow rate of influent; R and r is the external and inter-
2.5.1. Sludge sample
Triplicate sludge samples were collected from the aerobic zones
of the last chamber on days 18, 34, 56, 72, 100, 116, 154, 168 and
180. MLVSS concentrations were determined on the day of sam-
pling. Approximately 2 ml of mixed liquid was washed twice, i.e.
centrifuged it and then resuspended the pellet in 3  PBS, and then
centrifuged at 14,000g for 2 min. The supernatant was removed,
and the pellet was stored at 20 °C (Limpiyakorn et al., 2005).
2.5.2. AOB cultures
Genomic DNA extracted from enriched AOB communities was
used as standard DNA for real-time PCR analysis. The AOB-en-
riched culture was obtained by transfer of activated sludge with
good nitrification into a 300-ml conical flask and cultivation in
batch mode at 26 °C and agitation at 140 rpm. The medium con-
tained, per liter: 2 g (NH4)2SO4; 1 g K2HPO4; 0.5 g MgSO47H2O;
3 g NaHCO3; 0.4 g FeSO4; and 2 g NaCl. After 4 weeks, the nitrite
accumulation rate was steady at above 98% with a stable ammonia
removal of over 97%. Fluorescent in situ hybridization (FISH) anal-
ysis showed that AOB represented more than 80% of the culture
population.
2.5.3. DNA extraction
Genomic DNA was extracted in triplicate from 2 ml of MLSS
samples and enriched AOB cultures using Fast DNA SPIN kits for
soil (Bio 101, Vista, CA, USA). To minimize variations in DNA
extraction, the three extracts were mixed before the DNA was ana-
lyzed (Limpiyakorn et al., 2005).
2.5.4. Primer and probe
The AOB-specific PCR primers were selected to amplify a 116-
ð1Þ
bp DNA fragment in the V2 region of the 16S rDNA. Real-time
PCR quantification of 16S rDNA genes of total AOB was performed
using two forward primers CTO 189fA/B and CTO 189fC at a 2:1 ra-
ð2Þ tio, one reverse primer RT1r, and the Taq Man probe TMP1. The oli-
gonucleotide sequences of the primers and the Taq Man probe are
shown in Table 3 (Harms et al., 2003; Kindaichi et al., 2006).
ð3Þ
2.5.5. Standard curve for 16S rDNA quantification of AOB
The extracted DNA from enriched AOB culture was 10-fold di-
luted in sterile water and PCR was conducted in a 50-ll reaction

Table 2
Experimental scheme for partial nitrification in A2O process treating domestic wastewater.

Phase Influent flow rate Flow rate of internal Flow rate of sludge HRT (h) Anoxic HRT (h) Aerobic Anoxic AHRT (h) Aerobic DO
Q (L/h) recycle Qr (L/h) recycle QR (L/h) HRT (h) AHRT (h) (mg/L)
1 (1–16d) 7.83 23.49 6.26 9.31 2.54 4.97 0.53 1.03 1.5–2.5
2 (17–36d) 7.83 23.49 6.26 9.31 2.54 4.97 0.53 1.03 1.5–2.5
3 (37–58d) 5.22 20.88 5.22 13.96 3.82 7.45 0.64 1.24 0.1–0.3
(59–76d) 7.83 23.49 6.26 9.31 2.54 4.97 0.53 1.03 0.3–0.5
4 (77–104d) 7.83 31.32 6.26 9.31 2.54 4.97 0.44 0.86 0.3–0.5
(105–116d) 7.83 23.49 6.26 9.31 2.54 4.97 0.53 1.03 0.3–0.5
(117–124d) 6.26 26.36 5.01 11.63 3.18 6.21 0.53 1.03 0.3–0.5
5 (125–164d) 5.22 28.19 4.18 13.96 3.82 7.45 0.53 1.03 0.3–0.5
6 (165–180d) 5.22 15.66 4.18 13.96 3.82 7.45 0.80 1.55 0.3–0.5
 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082 8077

Table 3
The oligonucleotide sequences of the primers and the Taq Man probe.
Primer or Sequence (50 –30 )
probe
CTO 189fA/B GGAGRAAAGCAGGGGATCG
CTO 189fC GGAGGAAAGTAGGGGATCG
RT1r CGTCCTCTCAGACCARCTACTG
TMP1 (50 -FAM and 30 -TAMRA)b
CAACTAGCTAATCAGRCATCRGCCGCTC
a
E. coli numbering.
b 3.1. Achieving nitritation by low DO and short aerobic actual hydraulic
FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-tetramethylrhodamine.
mixture using a PCR kit (TaKaRa Ex Taq) containing 5 ll 10  EX
Taq buffer (Mg2+ plus), 4 ll dNTP (2.5 mM), 1 ll forward primer
CTO 189fA/B and CTO 189fC (10 lmol/L), 1 ll reverse primer
RT1r (10 lmol/L) (Harms et al., 2003; Kindaichi et al., 2006),
0.25 ll TaKaRa EX Taq (5 U/ll), 1 ll DNA template, and 37.75 ll
ddH2O. The program used for PCR amplification was as follows:
3 min at 94 °C, followed by 30 cycles consisting of 45 s at 95 °C,
30 s at 58 °C, and 45 s at 72 °C and a final cycle consisting of
4 min at 72 °C. The PCR products were visualized after electropho-
resis in 3% agarose. Agarose gel slices containing 116-bp DNA
bands were excised and the DNA was recovered and purified using
the TaKaRa Agarose Gel DNA Purification Kit Ver.2.0. The purified
target DNA was used as templates for a second round of PCR ream-
plification, and the resulting products were purified as before. The
concentration of the resulting DNA was measured with a spectro-
photometer, and DNA copy numbers were calculated.
Ten-fold serial dilutions of DNA of known copy numbers were
used as standard DNA. Each of the dilutions was real-time PCR
quantified in triplicate (Limpiyakorn et al., 2005). The real-time
PCR mixture was prepared in a total volume of 25 ll using the
TaKaRa Premix Ex Taq kit, containing 12.5 ll 10 Ex Taq Buffer
(Mg2+ Plus); 0.75 ll forward primer CTO 189fA/B and CTO 189fC
(10 lmol/L); 0.75 ll reverse primer RT1r (10 lmol/L); 1 ll TMP1;
1 ll standard DNA; 0.5 ll ROX Reference Dye II; and 8.5 ll ddH2O.
The real-time PCR amplification was performed in a Stratagene Mx
3005 instrument under conditions of 30 s at 95 °C, followed by 40
cycles of 5 s at 95 °C and 20 s at 60 °C.
2.5.6. Application of real-time PCR assays to MLSS samples
Three replicates per MLSS sample were analyzed using real-
time PCR. The real-time PCR mixture and amplifying conditions
for MLSS samples were the same as for the DNA standards. The
number of AOB cells per gram MLVSS was calculated based on
Positions (bp) comparison with the DNA standards and the assumption that
AOB contain one copy 16S rDNA as determined for the AOB Nitros-
189–207a omonas and Nitrosospira (Harms et al., 2003).
189–207a
283–304a
226–253a
3. Results and discussion
retention time (AHRT) control
An overview of nitrite accumulation rates, NO 
2 -N and NO3 -N
concentrations in effluent of the last aerobic zone is given in
Fig. 2. Fig. 3 presents the TN and ammonia removal throughout
the experimental period. Total, ammonia, nitrite and nitrate N con-
centrations along the reactor in the different phases were also
measured to gain a better insight into nitrogen removal (Fig. 4).
In phases 1 and 2, the A2O system was operated at a normal DO
concentration of 1.5–2.5 mg/L and aerobic AHRT of 1.03 h [calcu-
lated as formula (1)]. In comparison to the operation in phase 1,
the difference in phase 2 was that NaAc was supplied as external
carbon source for denitrification to enhance TN removal. Based
on the seed sludge with complete nitrification–denitrification,
the nitrite accumulation rate was almost zero during phases 1–2
(36 d) (Fig. 2). The experimental purpose of phase 3 (37–76 d)
was to investigate if nitritation could be achieved only by control
of low DO concentrations. In the initiation of phase 3, from day
37 to day 58, low DO condition (0.1–0.3 mg/L) was applied to sup-
press or eliminate NOB through expanding the differences of spe-
cific growth rates between AOB and NOB under limited aeration
(Blackburne et al., 2008); however, nitrite build-up did not occur,
and ammonia removal was only 30% (Figs. 2 and 3). It seems that
very low DO concentration (0.1–0.3 mg/L) limited AOB to oxidize
ammonia to nitrite to some extent, whereas a little longer aerobic
AHRT (1.24 h) was enough for NOB to oxidize nitrite to nitrate,
which resulted in a lower efficiency of ammonia removal and ni-
trite build-up. In the remaining 18 d of phase 3, at a slightly higher
DO level of 0.3–0.5 mg/L, nitrite build-up was not observed. There-
fore, nitritation could not be established only by control of low DO
concentrations.
Since low DO control failed to initiate nitritation, aerobic
AHRT was used as a selection factor. DO was kept constant at

- - - -
NO 3 -N NO 2 -N NO 2 -N/NO x -N
40 120
Ι ΙΙ ΙΙΙ ΙV V VΙ

100
30
NO2 -N NOx -N (mg/L)

80
NO2 -N/NOx -N (%)

20 60
-
-

40
-

10
-

20

0
0

0 20 40 60 80 100 120 140 160 180

Time (day)

Fig. 2. Nitrite accumulation rates during phase 1–6.

8078 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082

120 120
Ι ΙΙ ΙΙΙ ΙV V VΙ

NH 4 -N removal efficiency
TN removal efficiency (%)
100
100
80

(%)
60 80

40 60
20 ΤΝ

+
+ 40
0 NH4 -N
+
NH4 -N in influent
80
in influent and effluent

+ 6
NH4 -N in effluent

C/N in influent
60
NH 4 -N

(mg/L)

C/N 4
+

40

20 2

0
0
0 20 40 60 80 100 120 140 160 180
Time (day)

Fig. 3. Total and ammonia N removal in A2O process.

+ - -
TN NH4 -N NO2 -N NO3 -N
80 80
C/N=3.15 C/N=5.79
70 70
Nitrogenous compound (mg/L)

Nitrogenous compound (mg/L)

TN removal efficiency =60.9% TN removal efficiency=77.3%

+ +
60 NH 4 -N removal efficiency =99.1% 60 NH 4 -N removal efficiency=100%
50 50

40 40
30 30

20 20
10 10

0 0
inf pre An An An Ae Ae eff inf pre An An An Ae Ae eff
-an a o1 o2 ro1 ro2 -an a o1 o2 ro1 ro2
o o
(A)12d (phase Ⅰ) (B)32d (phase Ⅱ)
80 80
C/N=2.09 C/N=3.06
70 70
Nitrogenous compound (mg/L)

Nitrogenous compound (mg/L)

TN removal efficiency=77.6% TN removal efficiency=87.4%

+ +
60 NH 4 -N removal efficiency=99.5% 60 NH 4 -N removal efficiency=98.1%

50 50

40 40

30 30

20 20

10 10

0 0
inf pre A An An Ae Ae eff inf pre An An An Ae Ae eff
-an na o1 o2 ro1 ro2 -an a o1 o2 ro1 ro2
o o
(C)144d (phase Ⅴ) (D)168d (phase Ⅵ)

Fig. 4. Total, ammonia, nitrite and nitrate N concentrations along the reactor in A2O process (inf: influent; pre-ano: pre-anoxic zone; Ana: anaerobic zone; Ano1: the first
anoxic zone; Ano2: the second anoxic zone; Aero1: the first aerobic zone; Aero2: the second aerobic zone; eff: effluent).
 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082
0.3–0.5 mg/L, and during initiation of phase 4 (day 77–104), aerobic
AHRT was reduced to 0.86 h by increasing the flow rate of the
internal recycle (Table 2). Nitrite accumulation rates were gradually
increased and finally reached more than 80%, which implied that
NOB activities were successfully suppressed (Fig. 2); however, the
short aerobic AHRT resulted in poor ammonia removal. Thus, in
the middle of phase 4 (day 105–116), aerobic AHRT was slightly
increased to 1.03 h to improve ammonia removal, during which
the nitrite accumulation rate still remained above 80% (Fig. 2).
Thereafter, till the end of phase 5, with a constant aerobic AHRT
of 1.03 h, HRT in the system was increased to 11.63 h and 13.96 h,
respectively, to enhance TN removal through increasing the flow
rates of internal recycle as well as decreasing the influent flow rates.
During this period, the nitrite accumulation rate stabilized at over
90% with ammonia removal of above 95%. This outcome was possi-
bly due to suppression or reduction of NOB through low DO com-
bined with short aerobic AHRT control.
In order to investigate its impact on nitritation, aerobic AHRT
was extended to 1.55 h in phase 6. An obvious decline of nitrite
accumulation rate was observed, and the minimum was as low
as 33.6% (Fig. 2). These observations demonstrated that control of
aerobic AHRT was necessary to stabilize nitritation.
3.2. Mechanisms of nitritation in A2O system
In phases 4–5 (nitritation established and maintained), reactor
temperature in the reactor was controlled at 22 ± 1 °C and pH var-
ied from 7.2 to 7.6. This temperature and pH range could not be
used as selection factors to expand the differences of specific
growth rates between AOB and NOB. Since the average FA concen-
tration in the aerobic zones calculated with formula (4) was below
1 mg/L, this relatively low FA level (<1 mg/L) does not inhibit NOB
(Abeling and Seyfried, 1992; Peng et al., 2008). Therefore, temper-
ature, pH and FA concentration were unlikely factors contributing
to nitritation, in contrast to low DO concentrations, aerobic HRT
and AHRT.
3.2.1. Low DO concentration
The oxygen affinity constant of AOB and NOB is 0.3–0.5 mg/L
and 0.7–1.8 mg/L, respectively, indicating AOB having a stronger
300
Ⅰ Ⅱ
250
200
SVI (ml/g)
150
100
50
0 0.0
0 20 40
Fig. 5. Variations of SVI during phase 1 to 6.
8079
affinity with DO than NOB (Guisasola et al., 2005). Especially at
low DO concentrations, the specific growth rate of AOB is higher
than that of NOB (Blackburne et al., 2008). In this study, NOB activ-
ities were selectively inhibited under a long-term operation with
low DO levels of 0.3–0.5 mg/L; whereas AOB dominance was en-
hanced, leading to a nitrite build-up.
3.2.2. Aerobic HRT
Previous studies demonstrated that extension of aeration time
would result in a decline of nitrite accumulation rates (Peng
et al., 2007; Ma et al., 2009). Gao and Zeng et al. suggested that
control of aeration time in SBR process was a necessary method
to wash out NOB (Gao et al., 2009; Zeng et al., 2009). In this study,
during start-up period of nitritation in the A2O process, a short aer-
obic HRT (4.97 h) was applied to avoid excessive aeration and inhi-
bit NOB growth. In the initiation of phase 4 (day 77–104), ammonia
loadings were improved by increasing the flow rate of influent,
which favored AOB growth even though ammonia removal de-
clined. When nitrite accumulation rate stabilized at over 80%, aer-
obic HRT was gradually extended from 4.97 to 6.21 h and 7.45 h to
improve ammonia removal (Table 2).
3.2.3. Aerobic AHRT
Turk and Mavinic (1986) observed a lag time in nitratation
when changing from anoxic to aerobic condition, resulting in a
temporary, but significant, accumulation of nitrite which lasted
for several hours in the aerobic cell. Duration of aeration time
was inversely related to the degree of nitrite build-up, possibly
due to a lack of enzyme synthesis related to aerobic respiration
in NOB. Oxygen accepts electrons to form the reduction products
such as superoxide radicals, hydrogen peroxide and hydroxyl rad-
icals, which are extremely toxic. NOB are able to synthesize super-
oxide dismutase (SOD), catalase and peroxidase, which afford
protection against the above toxic intermediates through catalyz-
ing their destruction (Turk and Mavinic, 1986). At short aerobic
AHRT and high internal recycle, a quick alternating of anoxic/aero-
bic conditions did not allow enough time for NOB to synthesize the
necessary protective enzymes, leading to inhibition of activities.
In the continuous-flow A2O process, compared with aerobic
HRT, aerobic AHRT was more significant in establishing nitritation
SVI The average DO
3.0
Ⅲ Ⅳ Ⅴ Ⅵ
2.5
2.0
DO (mg/L)
1.5
1.0
0.5
60 80 100 120 140 160 180
Time (day)
8080 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082

than aerobic HRT. Even though aerobic HRT in phase 3 was the NO3 -N concentration below 18 mg/L on average. In phases 3–4,
same as that in the initiation of phase 4, nitrite build-up did not oc- ammonia and TN removal were reduced to 51% and 42%, respec-
cur in phase 3 (Fig. 2). During phases 4 and 5, nitritation was suc- tively, due to a low DO level and short HRT. In phase 5, due to a sta-
cessfully achieved through increasing the flow rate of internal ble performance of nitritation with a nitrite accumulation rate of
recycle to decrease aerobic AHRT. NO 2 -N generated in the aerobic above 94% (Fig. 2), the accumulated nitrite in the aerobic zone
zones was reduced in time due to quick recirculation of mixed li- was directly reduced to N2 in the anoxic zone through internal re-
quid (Figs. 3 and 4). The short aerobic AHRT was not long enough cycle. Therefore, TN was removed by the nitrite pathway in this
for NOB to further oxidize nitrite to nitrate in the aerobic zones. phase. Even though the average C/N ratio was only 2.3, TN removal
Therefore, NOB could not fully obtain nitrite substrate and their reached 75.4%, and ammonia removal was also increased to above
activities were suppressed at low DO levels. In phase 6, despite 95% through slightly extending HRT. At a lower C/N ratio, in com-
of low DO operation, nitritation did not occur due to increase of parison to phase 1 with TN removal by the nitrate pathway, TN re-
aerobic AHRT. This outcome further demonstrated the significance moval in phase 5 was 16% higher by the nitrite pathway, and was
of aerobic AHRT to nitritation. essentially equal to that of phase 2 with the addition of the exter-
nal carbon sources. In phase 6, the system changed from nitritation
3.3. Nitrogen removal by the pathway of nitrite and nitrate to complete nitrification as a result of extension of aerobic AHRT.
The addition of external carbon source in this phase raised TN re-
As shown in Figs. 2–4, in phase 1, at DO concentration of 1.5– moval to 85% with effluent TN concentration of only 9 mg/L on
2.5 mg/L, ammonia removal was above 95%, whereas due to a low- average. TN removal by the nitrite pathway at low DO levels of
er average C/N ratio (about 3) in influent, TN removal was only 60% 0.3–0.5 mg/L was essentially equal to that by the nitrate pathway
with an effluent NO 3 -N concentration up to 25 mg/L. In phase 2,
at DO levels of 1.5–2.5 mg/L with the addition of external carbon
NaAc was added as external carbon source to increase influent source. Therefore, TN removal by the nitrite pathway is highly ben-
C/N ratio to 5.9, and thus TN removal reached 74.4% with effluent eficial for domestic wastewater treatment with limited carbon

40

35 2
y = -3.341x+43.63 ; R = 0.999
Threshold cycles (Ct)

30

25

20

15

10
2 3 4 5 6 7 8 9 10 11
log10(copy number of standard DNA)
(A) Standard curve forreal-time PCR assays

6.00E+010 100
Numbers of AOB
Nitrite accumulation rate
Numbers of AOB (cells/g·MLVSS)

5.00E+010
80
Nitrite accumulation rate (%)

4.00E+010
60
3.00E+010
40
2.00E+010

20
1.00E+010

0.00E+000 0
18 34 56 72 100 116 154 168 180
Time (day)
(B) AOB quantification in sludge samples

Fig. 6. AOB quantification results using real-time PCR during phase 1–6.
 W. Zeng et al. / Bioresource Technology 101 (2010) 8074–8082
source in terms of lower carbon requirements and reduced aera-
tion costs.
3.4. Effect of low DO concentration on sludge volume index (SVI)
In a continuous-flow system, sludge bulking readily occurs dur-
ing long-term operation with low DO concentrations (Ma et al.,
2009). This occurrence influences sludge settling in the secondary
clarifier and is the main cause of unsatisfactory effluent quality
(Ma et al., 2009). However, in this study, sludge bulking was not
observed (SVI 100–120 ml/g) during the performance of nitritation
at low DO conditions (Fig. 5).
In phase 1, at normal DO concentrations of 1.5–2.5 mg/L, SVI
maintained at about 100 ml/g indicating a well-settling sludge. In
phase 2, with NaAc addition as external carbon source, sludge bul-
king occurred with SVI more than 200 ml/g (Fig. 5). A high amount
of filamentous organisms were observed that extended from the
flocs into the bulk solution and interfered with compaction and
settling. SVI rapidly rose over 200 ml/g. We hypothesized that
the addition of a readily biodegradable carbon sources (NaAc)
was the main cause of sludge bulking. In the initiation of phase 3
(days 37–58), at DO levels of 0.1–0.3 mg/L, SVI was still in the
range of 210–260 ml/g; however, from the middle of phase 3, with
DO at 0.3–0.5 mg/L, sludge settleability was obviously improved
and SVI gradually descended. Especially in phase 5, SVI stabilized
below 100 ml/g and nitritation–denitritation occurred. Therefore,
in this study, low DO (0.3–0.5 mg/L) operation did not result in
sludge bulking; on the contrary, settling characteristics improved.
In phase 6, even though external carbon sources (NaAc) were
added, sludge bulking did not occur and SVI stabilized at about
100 ml/g. NaAc dosage of COD 50 mg/L was depleted in the anaer-
obic and anoxic zones, and thus in the aerobic zones substrate was
not available to filamentous organisms. The above experimental
results are different from those of previous studies, in which SVI
variations showed a clear correlation with nitrite accumulation
rates (Casey et al., 1994; Ma et al., 2009). The mechanisms of better
sludge settleability at low DO levels and high nitrite accumulation
rate requires further investigation.
3.5. Dynamics of ammonia oxidizing bacteria (AOB) population
The DNA concentration obtained by PCR reamplification was
14.1 ng/ll measured with a spectrophotometer. This value was
converted to a DNA copy number of 1.03  1011 copies/ll. Serial
10 fold dilutions of DNA with known copy numbers were used
to generate a standard curve (y = 3.341x + 43.63), which shows
the relationship between DNA copy numbers and threshold cycle
(Ct) values (Fig. 6A). Copies numbers of standard DNA were highly
correlated with Ct values and demonstrated a linear relationship
with a regression coefficient (R2) of 0.999 and a slope of 3.341.
This outcome indicated that the standard curve was reliable for
quantification of AOB 16S rDNA.
The variations of AOB population throughout the experimental
period were quantified using real-time PCR (Fig. 6B). As shown in
Fig. 6B, AOB population had a clear correlation with nitrite accu-
mulation rates. In phases 1–2, at normal DO levels, the A2O system
showed very good complete nitrification, during which the AOB
population increased slightly from 4.66  109 cells/g MLVSS (on
day 18) to 5.28  109 cells/g MLVSS (on day 34). Subsequently,
nitrification deteriorated due to DO decline, leading to a decrease
in AOB by 33.5% on day 56. In phase 4, nitrite accumulation rates
gradually increased as aerobic AHRT was reduced. On day 100,
the AOB population reached 4.40  1010 cells/g MLVSS correspond-
ing to a nitrite accumulation rate of 84.2%. During the subsequent
time till to the end of phase 5, the AOB population stabilized at
about 4  1010 cells/g MLVSS, meanwhile nitrite accumulation
8081
rates maintained at 90% on average. These results indicate that
the AOB population tended to be stable during steady nitritation.
In phase 6, nitrite accumulation rates decreased from 85.2% to
33.6% due to the extension of aerobic AHRT. Correspondingly,
AOB populations were reduced to 2.18  1010 cells/g MLVSS on
day 168.
4. Conclusions
Achievement of nitritation–denitritation is highly beneficial for
the treatment of domestic wastewater with limited carbon sources
in terms of lower carbon requirements and reduced aeration costs.
A combination of short aerobic AHRT and low DO concentrations
was an effective strategy to achieve nitritation in a continuous-
flow A2O process treating domestic wastewater at ambient
temperatures. Real-time PCR assays demonstrated that low DO
concentration combined with short aerobic AHRT promoted AOB
growth. Low DO did not result in deterioration of sludge
settleability.
Acknowledgements
This work was financially supported by the Natural Science
Foundation of China (No. 50878005), Beijing Natural Science Foun-
dation (No. 8102005) and Fok Ying Dong Education Foundation
(No. 121076).
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