Separation and Purication Technology 63 (2008) 670675

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Separation and Purication Technology

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Performance of a nitrifying airlift reactor using granular sludge

Ren-Cun Jin a,b , Ping Zheng a, , Qaisar Mahmood a , Lei Zhang a
a
b

Department of Environmental Engineering, Zhejiang University, Hangzhou 310029, PR China

Department of Environmental Science, Hangzhou Normal University, Hangzhou 310036, PR China

a r t i c l e

i n f o

Article history:
Received 19 November 2007
Received in revised form 12 July 2008
Accepted 14 July 2008
Keywords:
Airlift reactor
Granular sludge
Nitrication
Anaerobic ammonium oxidation

a b s t r a c t
Since nitrication is the rate-limiting step in the biological nitrogen removal from wastewater, many studies have been conducted on the immobilization of nitrifying bacteria. A laboratory-scale investigation was
conducted to examine the effectiveness of a continuous-ow airlift reactor (ALR) on the granulation of
nitrifying sludge and the nitrication efciency of the reactor after granulation. The results showed that
the granular sludge began to appear on day 30 and matured in 75 days. The mature granules had an average
diameter of 1.54 mm, settling velocity higher than 82.4 m h1 and specic gravity of 1.07. The granules cultured in the present study had aerobic ammonia oxidation activity of 13.3 mg NH4 + -N (g VSS)1 day1 and
anaerobic ammonium oxidation (ANAMMOX) activity of 3.22 mg NH4 + -N (g VSS)1 day1 , which demonstrated that the nitrifying granules possessed the potential to be used as seed sludge for ANAMMOX and
CANON (completely autotrophic nitrogen-removal over nitrite) reactors. After granulation, the ALR exhibited an excellent nitrication performance. It had strong tolerance to inuent NH4 + -N of 1100 mg L1 .
When operated at inuent NH4 + -N concentration of 546 mg L1 , the reactor could remove over 94.4%
of ammonium even at a nitrogen loading rate (NLR) of 2.37 f m3 day1 with a short hydraulic retention time (HRT) of 5.4 h. With the inuent NH4 + -N concentration of 547 mg L1 , HRT 6.9 h and NLR of
1.90 kg m3 day1 , superior efuent quality could be achieved robustly, with an efuent NH4 + -N of less
than 5 mg L1 , satisfying the national primary discharging standard of China (GB 8978-1996).
2008 Elsevier B.V. All rights reserved.

1. Introduction
Nitrogenous compounds like ammonium are prevalent in many
wastewaters and need to be removed to prevent oxygen depletion and eutrophication of surface waters. Biological nitrogen
removal from wastewater using nitricationdenitrication is a
well-known and cost-effective treatment process [1,2]. Because of
extremely low growth rate, it is generally accepted that retaining a
large number of nitrifying bacteria within the reactor is difcult to
achieve, thereby making the nitrication a rate-limiting step in the
entire nitrogen removal process [2,3].
Many researches have been carried out to develop the physical
or ecological methods of immobilizing nitrifying bacteria including
cell-entrapping and cell-attaching techniques [46]. However, the
immobilized cells created by gel entrapping are easy to be suffered
from mass transfer resistance [6]. Previous researches also demonstrated that construction of a nitrifying biolm on the surface of
carrier materials takes a long time, particularly when the wastewater contains few organic compounds [7]. Moreover, the matrices
and carriers used for cell immobilization inevitably occupy sig-

Corresponding author. Tel.: +86 571 86971709; fax: +86 571 86971709.
E-mail address: pzheng@zju.edu.cn (P. Zheng).
1383-5866/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2008.07.012

nicant space in the reactor, limiting cell density. To avoid such

problems, granular sludge was generated to enhance cell retention
and biomass concentration, simultaneously.
Sludge granulation has been intensively studied in anaerobic
systems such as upow anaerobic sludge blanket (UASB) [8] and
in aerobic systems [912], but these reports have mostly focused
on organic pollutant removal and the aerobic granular reactor used
in these tests was mainly sequencing batch reactor (SBR) [9,11,12].
Until now, fewer attempts have been made to culture granular
sludge in a continuous-ow system.
Three-phase airlift reactor (ALR) is being applied frequently in
chemical, biotechnological and environmental processes as simple
and effective gasliquidsolid phase contactor. ALR offers advantages over traditional three-phase contactors, namely, a lower gas
requirement for complete suspension of the solid, elimination of
dead volumes, rapid mixing and the absence of external recirculation systems [1315].
In this study, a continuous-ow ALR was used to culture nitrifying granular sludge. The ALR can be operated at a lower aeration
volume than other types of aerobic bioreactors; therefore, ocs of
nitrifying bacteria in the ALR are expected not to crumble and form
granules. The techniques for the granulation of nitrifying bacteria
in the ALR and the nitrication performance of the reactor with
granular sludge are reported in the present paper.

R.-C. Jin et al. / Separation and Purication Technology 63 (2008) 670675

671

equivalent diameter of 100 granules randomly obtained from the

ALR after every 15 days. Measurements of specic gravity and settling velocity of granular sludge were performed using the methods
as described by Zheng et al. [17]. The aerobic ammonia oxidation
activity and anaerobic ammonium oxidation (ANAMMOX) activity
of sludge was determined by batch test as described by Windey et
al. [18].
2.5. SEM observation

2. Materials and methods

Physiological characteristics of the biomass were observed using

SEM model PHILIPS XL30ESM. The specimens for SEM were prepared by xing the biomass with 2.5% glutaraldehyde in 0.1 mol L1
phosphate buffer at pH 7.2 for 2 h. After that, the specimens were
rinsed twice in a buffer for 10 min each time and once in distilled
water for 5 min, followed by dehydration with a graded series of
ethanol (50%, 70%, 80%, 90% and 95%), and dried at critical point,
then mounted and coated.

2.1. ALR

3. Results and discussion

The schematic diagram of the internal-loop ALR used in the

present study is shown in Fig. 1. This reactor was made of perspex with a working volume of 9.2 L, consisting of four sections:
riser, downcomer, gas separator, and settling section. Aeration was
done at the bottom of the reactor via a porous air diffuser ball with
an air ow rate of 13.7 L min1 to keep the dissolved oxygen (DO)
concentration in both riser and downcomer above 5.0 mg L1 . The
reactor was operated at pH range of 7.58.5 and at 30 1 C in a
room equipped with thermostat.

3.1. Formation and characteristics of granules

Fig. 1. Schematic illustration of the ALR used in the experiment: (1) inuent tank, (2)
peristaltic pump, (3) efuent tank, (4) riser, (5) downcomer, (6) settler, (7) overow
weir, (8) air pump.

2.2. Inoculum
Aerobic sludge from a local municipal wastewater treatment
plant was used as inoculum for the present study and the initial
volatile suspended solid (VSS) concentration was 10 g L1 .
2.3. Synthetic wastewater
The composition of synthetic wastewater is listed in Table 1. For
alkalinity and carbon source supplement, the theoretical NaHCO3
requirement for nitrication (7.1 g as CaCO3 (g NH4 + -N)1 ) was
added to the wastewater.
2.4. Analytical procedures
Ammonium, nitrite, nitrate, VSS and pH were determined using
the standard methods [16]. DO was measured by JPB-607 dissolved
oxygen meter. The average diameter of granules was measured
using an optical microscope (YS2-H, Nikon, China) as a circleTable 1
Composition of synthetic ammonium-containing wastewater
Compound

Concentration

KH2 PO4
MgSO4 7H2 O
CaCl2
NaHCO3
(NH4 )2 SO4
Trace elements Ia
Trace elements IIb

0.027 g L1
0.300 g L1
0.136 g L1
0.8413.3 g L1
0.335.23 g L1
1.25 mL L1
1.25 mL L1

Composition of trace elements I (g L1 ): EDTA 5.00, FeSO4 5.00.

Composition of trace elements II (g L1 ): EDTA 15, ZnSO4 7H2 O 0.43, CoCl2 6H2 O
0.24, MnCl2 4H2 O 0.99, CuSO4 5H2 O 0.25, Na2 MoO4 2H2 O 0.22, NiCl2 6H2 O 0.19,
Na2 SeO4 10H2 O 0.21, H3 BO4 7H2 O 0.014.
a

The process for granulation of nitrifying sludge lasted for 75 days

and completed in three phases: seed sludge acclimation, occulation and granulation. After acclimation for 15 days, the dispersed
seed sludge (Fig. 2a) self-aggregated to form ocks (Fig. 2b). Granulation of sludge occurred within 15 days thereafter (Fig. 2c), but
the granules were not dense enough. Subsequently, the working
hydraulic retention time (HRT) of the reactor was shortened to produce a higher hydraulic selection pressure, inducing formation of
larger and denser granules [19]. Till day 75, the granular sludge
with high activity and sedimentation ability prevailed in the ALR.
The SEMs of the granules sampled from the ALR on day 75 are presented in Fig. 3. It was conrmed that the bacteria formed dense
layers on the granular surfaces.
It was reported that granules in UASB possess lamentous
bacteria on their outermost layer [20]. The lamentous bacteria
were also present on the surfaces of the nitrifying granules, which
disagreed with the results of Tsuneda et al. [10], which might
be due to the differences in characteristics of seed sludge. It is
assumed that the self-aggregation of microbes is characteristic to
methane- and hydrogen-producing sludge [21] and even to denitrifying sludge [22]. Because these bacteria were heterotrophic,
they produced more extracellular polysaccharides compared with
autotrophic bacteria. Because of this property, heterotrophic bacteria were expected to be superior to autotrophic ones in terms of
granulation. From this standpoint, it is difcult to develop nitrifying
granular sludge as observed in the present study.
De Beers research group cultured nitrifying granules in inorganic wastewater using a pre-aeration system where shear stress
on sludge caused by bubbling was suppressed [2325]. On the other
hand, the present study demonstrated that nitrifying granules were
produced in spite of aeration in the reactor. It was inferred that
the role of air bubbles in the ALR corresponded to that of methane
gas bubbles in the UASB process. In addition, the ALR has a larger
gasliquid interface and a lower aeration volume than other three
phase systems. It was assumed that, due to this lower aeration volume; the ocs of nitrifying bacteria were not destroyed, thereby
promoting self-aggregation. Actually, the experiments performed
by Tsuneda et al. [26] with various aeration volumes revealed that
the granules were only produced if the aeration volume in the uidized bed was in the range of 0.0710.20 L min1 (L-bed)1 . However, when the aeration volume was set at 0.036 L min1 (L-bed)1 ,
granulation was not observed probably due to too low shear stress.

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R.-C. Jin et al. / Separation and Purication Technology 63 (2008) 670675

Fig. 2. Optical microscope photograph of nitrifying sludge: (A) seed sludge (10 10), (B) oc-like sludge (10 10), (C) granular sludge (10 10), (D) granular sludge (10 20).

From these results, a moderate aeration volume seems to promote

the nitrifying sludge granulation. This nding is also supported by
the report of Liu and Tay [27] who stated that hydrodynamic shear
force promotes the production of extracellular polymeric substrate,
which was assumed to play an important role in sludge granulation.
The spherical or elliptical nitrifying granules possessed coffee
color, with rough surfaces containing lamentous bacteria on their
surface layer (Fig. 2). The granular sludge could be stored in good
condition at 4 C in an anaerobic serum ask, without any obvious
damage for 6 months.
The granules ranged in size from 0.35 to 2.41 mm. The percentage of granules having diameter above 1.0 mm was 29% on day 30
that increased to 81% on day 75 (Table 2), suggesting an increase in
size with the passage of operational time.
As shown in Table 3, the setting velocity of granular nitrifying
sludge was 82.4 m h1 on day 75, higher than those reported earlier
[9,10], indicating that those particular nitrifying granules could be
Table 2
Distribution (%) of diameters of sludge granules
Time (day)

Diameter (mm)
<0.5

0.51.0

1.00.5

>1.5

30
45
60
75

22
2
0
3

49
24
27
16

23
45
35
34

6
29
38
47

Table 3
Physical characteristics of nitrifying granular sludge
Time(day)

Average diameter (mm)

Settling velocity (m h1 )
Specic gravity

30

45

60

75

0.82
55.5
1.01

1.30
75.6
1.08

1.40
74.9
1.05

1.54
82.4
1.07

effectively retained when used in a continuous stirring tank reactor under perfectly mixed-ow conditions at a short HRT of 5.4 h.
The excellent setting capability of granules in the present study
was assumed to be due to the seed sludge characteristics or/and
the operational conditions used which were still unclear and are
subjected to further clarication.
The granules possessed excellent ammonia oxidation activity.
The batch test results shown in Fig. 4 indicated that, under given
experimental conditions, the time taken for complete oxidation of
168 mg N L1 ammonium by nitrifying granules was 32 h, in contrast to 200 h for seed sludge. The aerobic ammonia oxidation
activity of nitrifying granules (13.3 mg NH4 + -N (g VSS)1 day1 )
was 5.8 times higher than that of seed sludge (1.96 mg NH4 + N (g VSS)1 day1 ).
The ANAMMOX activity was detected in nitrifying granules on
day 120, which indicated the presence of ANAMMOX bacteria. The
granules conducted ANAMMOX reaction at a rate of 3.22 mg NH4 + N (g VSS)1 day1 and the observed ratio of ammonia oxidation rate
to nitrite reduction rate was 0.96, near the stoichiometry of ANAMMOX. The ANAMMOX process is a biological reaction in which
ammonia is oxidized to nitrogen gas using nitrite as the electron
acceptor under anoxic conditions [28].
The ANAMMOX reaction appears to occur only under strict
anaerobic conditions. As little as 1.1 mM O2 was sufcient to
inhibit ANAMMOX activity completely in a bioreactor [29]. The
inhibition was reversible, and in experiments with intermittent aeration, the rate of ANAMMOX before and after aeration was identical.
The presence of ANAMMOX bacteria in granules detected in the
present study may be due to the layered structure of granules [10],
where the nitrifying bacteria in surface layer almost utilize all of the
oxygen and the resistance for oxygen mass transfer is high enough,
keeping a strict anaerobic environment for ANAMMOX organisms.
The ANAMMOX process should be combined with partial
nitrication stage, in which around 50% of ammonia could be
converted to nitrite. The process offers advantages for the treatment of efuents decient in organic matter, compared with the

R.-C. Jin et al. / Separation and Purication Technology 63 (2008) 670675

673

Fig. 4. Evolution of NH4 -N concentration in batch test for nitrifying activity.

due to the higher growth rate of aerobic sludge, the seed nitrifying
granular sludge can be harvested in large quantities subsequently.
The aerobic and anaerobic ammonium oxidizing bacteria could
coexist in granular sludge under investigation, even at a high dissolved oxygen level above 5.0 mg L1 . When the reactor is operated
under oxygen-limiting conditions, the ANAMMOX organisms are
able to grow and partial nitrication and ANAMMOX are engineered
in a single reactor. The process has been named CANON, an acronym
for completely autotrophic nitrogen-removal over nitrite [31]. In
this setup, aerobic ammonia-oxidizing bacteria consumed all the
oxygen, thus maintaining a very low oxygen concentration in the
granules and produced nitrite as the electron acceptor for ANAMMOX. In CANON process, the entire nitrogen removal can be carried
out in a single reactor with very low aeration, greatly reducing space
and energy requirements. Its feasibility, effectiveness and stability have been established by some studies, achieving a nitrogen
removal rate of 1.5 kg m3 day1 in a gaslift reactor [31], which is
more than sufcient to start application trials. The results in present
study suggest that CANON reactor could be started up directly by
seeding the nitrifying granules.
3.2. Performance of ALR
3.2.1. Performance during start-up
As shown in Fig. 5, the reactor was started at 24 h HRT and inuent NH4 + -N concentration of 70 mg L1 , corresponding to a nitrogen

Fig. 3. SEM (scanning electron microscope) photograph of nitrifying granules in the

airlift reactor at day 75. (A) 50, (B) 500, (C) 2500.

nitricationdenitrication process. This process allows the reduction in the oxygen requirements and carbon dioxide emission to the
atmosphere. However, the practical application of the ANAMMOX
process is still limited by its long start-up period due to the very
low growth rate (0.072 day1 measured at 32 C) of bacterial communities involved and biomass yield generated per gram ammonia
nitrogen consumed (0.088 g g1 ) by these microorganisms [30]. For
these reasons, the inoculum with high ANAMMOX activity is desirable, especially in situations where the industrial scale ANAMMOX
systems are operated. The present study suggested that the nitrifying granules were potential seed sludge of ANAMMOX reactor, and

Fig. 5. Evolution of the performance of nitrifying bioreactor during start-up.

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R.-C. Jin et al. / Separation and Purication Technology 63 (2008) 670675

Table 4
Performance of airlift reactor with different working HRTs
HRT (h)

22.8
19.5
15.9
13.1
11.8
9.61
9.01
7.38
7.02
6.34
6.25
6.02
5.41

NH4 + -N (mg L1 )
Inuent

Efuent

528
566
570
535
560
512
534
520
531
519
553
571
594

2.52
4.06
0.56
0
6.86
0.56
12.3
28.6
29.0
0.42
14.4
31.5
21.1

Removal efciency (%)

NLR (kg m3 day1 )

Ammonia removal rate

(kg N m3 day1 )

Efuent NO2 -N (mg L1 )

Efuent NO3 -N (mg L1 )

99.5
99.2
99.9
100
98.8
99.9
97.7
94.4
94.6
99.9
97.4
94.5
96.5

0.556
0.697
0.864
0.979
1.14
1.28
1.42
1.69
1.82
2.00
2.10
2.27
2.37

0.553
0.691
0.863
0.979
1.13
1.28
1.39
1.60
1.72
1.99
2.04
2.15
2.29

0
0
44.7
1.68
51.2
0
0
276
251
73.6
140
232
111

377
440
427
486
472
491
337
142
194
383
295
179
353

loading rate (NLR) of 0.07 kg m3 day1 . The activity of nitrifying

bacteria was stimulated by raising the inuent ammonium concentration at xed HRT. During the initial 7 days, the ALR exhibited
poor performance, with an average removal rate of 49.3% and a
mean ammonia removal rate of 0.084 kg N m3 day1 . In the next 8
days, the reactor capability improved signicantly and the removal
efciency enhanced to 100%. Upon increasing the inuent NH4 + -N
concentration from 76.6 to 774 mg L1 with a concomitant increase
of NLR to 0.774 kg m3 day1 , the removal became stable over 95%
during days 1645.
3.2.2. Performance of ALR at various inuent concentrations
After a satisfactory start-up, the reactor was operated until a
pseudo-steady-state was reached, as indicated by a constant efuent substrate concentration (5%). The experiment was conducted
to show the performance of ALR at different inuent concentrations. The inuent concentration was increased from 774 to
1110 mg L1 , keeping HRT xed at 24 h. Each run at one concentration lasted over 1 week before shifting to the next condition.
Data based on arithmetic means of three or more measurements
obtained at pseudo-steady-state are reported.
The ammonium removal efciencies of the reactor were near
100% when the inuent NH4 + -N concentration was in the range of
7741110 mg L1 , corresponding to NLR of 0.7741.11 kg m3 day1 ,
with nitrate as the main product while the efuent NO2 -N concentration was below 12.6 mg L1 . The results indicated that the
ALR could treat ammonium-containing high strength wastewater.
However, the performance of ALR went worse upon increasing the inuent NH4 + -N concentration over 1120 mg L1 , with
a removal efciency of 64.7%. Similarly, the efuent NO2 -N
increased to 52.9 mg L1 , resulting in nitrite accumulation that may
be ascribed to free ammonia inhibition.

3.2.3. Performance of ALR at various HRTs

In order to avoid the inhibition of high strength inuent substrate, the NLR was progressively increased by decreasing the
operating HRT, while keeping the inuent NH4 + -N constant at
546 mg L1 , to determine the maximum treatment capacity of the
ALR. The data presented here were the arithmetic means of three
or more measurements obtained at pseudo-steady-state.
As shown in Table 4, the HRT of ALR was gradually decreased
from 22.8 to 5.4 h during the HRT test, with a concomitant
increase in NLR from 0.56 to 2.37 kg m3 day1 , attaining the
NH4 + -N removal always higher than 94.4%. Thus the ammonia
removal rate reached 2.29 kg N m3 day1 , which was fourfolds
of 0.40.5 kg N m3 day1 achieved by the conventional nitrication process using activated sludge and nitrifying biolms [2,32].
Moreover, Table 5 shows that the ALR could be operated stably
at NLR of 1.90 kg m3 day1 , with removal efciencies above 99%
and efuent NH4 + -N concentration below 5 mg L1 , satisfying the
national primary discharging standard of China (GB 8978-1996)
[33]. The stepwise change in NLR might have resulted in efcient growth and retention of nitrifying bacteria in the granules
because the specic growth rate of each cell is governed by the
NLR per number of nitrifying bacterial cells (food to microorganism ratio, F/M). When the number of ammonia-oxidizing bacterial
cells increased in a granule with the passage of time, a higher
NLR was required to maintain their maximum specic growth rate
and to enhance granulation further. Results suggest that the use
of granules realizes the retention of large amounts of nitrifying
bacteria in the reactor, which guarantees highly efcient nitrication.
There was nitrogen loss in the ALR shown by mass balance
between inuent and efuent total nitrogen, achieving peak of
19.2% of inuent total nitrogen. The autotrophic process such as

Table 5
Performance of airlift reactor at inuent NH4 + -N of 547 mg L1 and HRT of 6.9 h
Time (day)

1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th

NH4 + -N (mg L1 )
Inuent

Efuent

524
567
546
542
560
567
528
558
508
570

2.38
4.76
4.48
4.76
3.92
2.80
0.14
0
0
0

Removal
efciency (%)

NLR (kg m3 day1 )

Ammonia removal rate

(kg N m3 day1 )

Efuent NO2 -N (mg L1 )

Efuent NO3 -N (mg L1 )

Nitrogen
loss (%)

99.6
99.2
99.2
99.1
99.3
99.5
100
100
100
100

1.94
1.89
2.03
1.96
1.85
1.90
1.79
1.91
1.83
1.93

1.89
1.83
1.97
1.90
1.80
1.85
1.75
1.87
1.79
1.89

450
404
368
389
343
187
155
86.4
69.9
98.1

68.7
54.2
115
100
179
328
319
365
434
452

0.6
18.4
10.9
8.9
6.1
8.8
10.4
19.2
0.9
3.5

R.-C. Jin et al. / Separation and Purication Technology 63 (2008) 670675

ANAMMOX as mentioned above may be a mean of nitrogen loss

from ALR, since inorganic wastewater is fed completely.
4. Conclusions
During the present study, the granulation of nitrifying bacteria
using an ALR was characterized and the nitrication performance
of the ALR with granules was investigated. The experimental results
are summarized as follows:
1. Nitrifying granules were observed in the ALR after operation of
30 days, and they matured in day 75. The mature granules had an
average diameter of 1.54 mm, settling velocity above 82.4 m h1
and specic gravity of 1.07.
2. The granules cultured in the present study had aerobic ammonia oxidation activity of 13.3 mg NH4 + -N (g VSS)1 day1 and
ANAMMOX activity of 3.22 mg NH4 + -N (g VSS)1 day1 , which
demonstrated that the nitrifying granules possessed the potential to be used as seed sludge for ANAMMOX and CANON reactors.
3. The continuous treatment of inorganic wastewater containing
546 mg L1 of NH4 + -N using the ALR with granules demonstrated
that the ammonia removal rate reached 2.29 kg N m3 day1 by
stepwise reduction in HRT to 5.4 h, suggesting that the nitrifying
granules enabled stable and efcient nitrication.
Acknowledgements
The authors wish to thank the National Hi-Tech Research and
Development Program of China (863) (2006AA06Z332), and the
Science and Technology Foundation for Key Project of Zhejiang
province (2003C13005) for the partial support of this study.
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