Trans. Tianjin Univ.

2013, 19: 255-259

DOI 10.1007/s12209-013-1966-z

CANON Process for Nitrogen Removal from Effluents

of Municipal Sewage Treatment Plants*
Peng Xinhong(彭新红)，Wu Libo(吴立波)，Yu Hongbing(于宏兵)，
Ai Lina(艾丽娜)，Fu Lixia(付丽霞)
(College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China)

© Tianjin University and Springer-Verlag Berlin Heidelberg 2013

Abstract：The feasibility and performance of nitrogen removal from municipal sewage were investigated through the
completely autotrophic nitrogen removal over nitrite (CANON) process in a continuous reactor. CANON process was
successfully started up with the transformation of nitrogen into gas by mass-balance analysis. For the synthetic waste-
water (up to 480 mg NH4+-N/(L·d)), removal rates of the ammonia nitrogen and total nitrogen (TN) were about
80% and 55%, respectively, at 1.25 h hydraulic retention time (HRT). For the secondary effluent of municipal sewage,
the effluent concentrations of NH4+-N and TN were below 5 mg/L and 9 mg/L, respectively. It is in accordance with
the water quality standard for scenic environment with the reuse of urban recycling water (GB/T 18921—2002).
Keywords：biodegradation; CANON; optimisation; municipal sewage

Nitrogen removal has become a major problem in requirements.

water quality management. Biological nitrification- Partial nitrification：
NH4++1.5O2→NO2 +2H++H2O
-
denitrification is the most commonly used process for (1)
nitrogen removal from wastewater, especially municipal Anammox:
wastewater[1]. However, complete nitrogen removal is - -
NH3+1.3NO2 →1.02N2+0.26NO3 +2H2O (2)
limited by the availability of reducing power in the form CANON:
of organic substrates. As an alternative to the costly addi- NH4++0.85O2→0.435N2+
0.13NO3 +0.14H++1.3H2O
-
tion of carbon substrates to anoxic reactors, the replace- (3)
ment of nitrate with nitrite[2] and the application of the With the Anammox bacteria found in the bottom of
anaerobic oxidation of ammonia (Anammox)[3] have Black Sea by Kuypers et al[6], Anammox process be-
been considered as novel nitrogen removal processes. comes promising for the treatment of low-ammonium
The Anammox process consists of the anaerobic oxida- content wastewater. In this study, a laboratory-scale bio-
tion of ammonia with nitrite as electron acceptor without reactor was operated to investigate the feasibility of ni-
additional organic carbon source[4]. This process pro- trogen removal by CANON process, and the influences
duces low excess sludge and requires only 40% of the of pH and temperature on the performance were taken
aeration energy compared with the conventional nitrogen into account.
removal process.
The Anammox process is usually performed in two 1 Materials and methods
ways: (1) single reactor system for high activity ammo-
nium removal over nitrite (Sharon)[2,4] in two continuous 1.1 Reactor description
units; (2) completely autotrophic nitrogen removal over The upflow biofilter reactor was constructed by the
nitrite (CANON) process in one unit[5], as shown in plastic (Plexiglas) cylinder of 27 mm internal diameter
Eqs. (1)—(3). The CANON process is more economi- (net volume of 150 mL with the total volume of 190
cal and environmentally friendly than the traditional ni- mL), as shown in Fig. 1, which was wrapped by a black
trogen removal systems, due to its lower cost and energy plastic in order to inhibit the effect of light on Anammox

Accepted date: 2012-10-19.

*Supported by National Natural Science Foundation of China (No. 50308012 and No.21107053); National Science & Technology Program of
China (No.2012ZX07501002).
Peng Xinhong, born in 1981, female, doctorate student.
Correspondence to Yu Hongbing, E-mail: hongbingyu1130@sina.com.cn.
 Transactions of Tianjin University Vol.19 No.4 2013
activity. The bioceramic with the diameter of 1—3 mm
was used as support materials for the biofilm. There were
three peristaltic pumps in Fig.1: one on the bottom as the
feeding solution inlet; another for maintaining a constant
operating temperature; and the third for making the efflu-
ent recycle with the reflux ratio of 10. PharMed tubing
(made of thermoplastic elastomer polypropylene) and
N2-filled gas bag connected to the influent tank were used
to minimize oxygen penetration.
1—Influent tank; 2—Metering pump; 3—Thermostated water tank;
4—Hot water recycle pump; 5—Reflux pump; 6—Gas outlet;
7—Effluent water.
Fig.1 Schematic diagram of the laboratory-scale reactor
1.2 Seed biomass
The reactors were initially inoculated using a pre-
acclimated cell suspension from an Anammox upflow
blanket filter reactor packed with ester non-woven bio-
mass carrier. The pre-acclimated cell suspension con-
tained a significant amount of Anammox biomass[7]. The
activity of Anammox bacteria was analyzed in standard
batch cultures (date not shown). Some physical and
chemical properties of Anammox sludge were as follows:
volatile suspended solid (VSS) 1.9 g/L, suspended solid
(SS) 4.43 g/L, VSS/SS 42.89%. The biomass with am-
monium-oxidizing bacteria (AOB) was obtained from
Jizhuangzi Wastewater Treatment Plant (WWTP) in
Tianjin, China.
1.3 Start-up and operation strategy
The reactors were operated under the constant tem-
perature of 32 ℃ in batch mode and started up with arti-
ficial synthetic media. The dissolved oxygen (DO) con-
centration was controlled at 0.02 mg/L by N2 gas bub-
bling. Organic carbon was not added to the synthetic
wastewater, as described in Ref.[8]. The composition was 2 Results and discussion
as follows: KHCO 3 0.5 g/L, KH 2 PO 4 0.271 2 g/L,
MgSO4·7H2O 0.3 g/L, CaCl2 0.136 g/L, trace element 2.1 Start-up CANON process
solutions Ⅰ and Ⅱ referred to Ref.[9], and NH4Cl and
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NaNO2 in required concentration. The trace element solu-
tion Ⅰ consisted of EDTA 5 000 mg/L and FeSO4
5 000 mg/L. The trace element solution Ⅱ consisted of
EDTA 5,000 mg/L, ZnSO4 · 7H2O 430 mg/L,
CoCl2 · 6H2O 240 mg/L, MnCl2 · 4H2O 990 mg/L,
CuSO4 ·5H2O 250 mg/L, Na2MoO4 ·2H2O 220 mg/L,
NiCl2·6H2O 190 mg/L, Na2SeO4·10H2O 210 mg/L and
H3BO4 14 mg/L. The pH of the feed was adjusted within
the range of 7.8—8.1 before it was added to the reactor.
Partial nitrification was introduced into the Anammox
process to start up CANON system. Then, the experiment
was divided into two parts: (1) examining the influences
of pH and temperature on the CANON process; (2)
evaluating the feasibility of treating the effluent of sec-
ondary sedimentation tank (Jizhuangzi WWTP, Tianjin,
China) with CANON process.
1.4 Analytical methods
VSS was measured by drying at 550 ℃ on an
evaporating dish, SS by drying at 105 ℃ on a 1 μm glass-
fiber filter, COD by microwave digestion method, NH4+-
N by Nessler’s reagent spectrophotometry (DR/4000U,
- -
HACH, US), and NO2 -N and NO3 -N by ion chroma-
tography, all of which were described in detail by stan-
dard methods[10]. The pH and DO were measured by a pH
meter (Mettler Toledo 320, Mettler-Toled, Switzerland)
and a DO meter (HQ-10, HACH, US), respectively.
1.5 Calculations
Nitrite accumulation ratio (NAR) was calculated by
chemical equilibrium[11]. TN was calculated as the sum of
concentrations of NH4+-N, NO2 -N and NO3 -N. Concen-
- -
trations of free ammonia (FA) and free nitrous acid
(FNA) were calculated as a function of pH, temperature
and total ammonia nitrogen(TAN), and a function of pH,
temperature and total nitrite (TNO2), respectively[8]:
NO2
NAR   100% (4)
NO2  NO3
TAN
FA(mg/L)   pH (5)
1  ( 10 / KeNH )
TN O2
FNA(mg /L)  (6)
1  ( KeNO / 10 pH )
KeNH  e 6 344 /( 273T ) (7)
2 300 /( 273  T )
NO
Ke  e (8)
In period A (1—46 d), Anammox process was run-
 Peng Xinhong et al: CANON Process for Nitrogen Removal from Effluents of Municipal Sewage Treatment Plants

ning without oxygen supply. In order to accumulate the
Anammox bacteria, the hydraulic retention time (HRT)
was gradually shortened to 1 h with the nitrogen loading
of 550 mg NH4+-N/(L·d), but the removal rates of both
NO2 -N and NH4+-N were below 80%. Therefore, the
-
HRT was back to 1.25 h with the nitrogen loading of 480
mg NH4+-N/(L·d), and the removal rates were above
90%, as shown in Fig. 2. Partial nitrification is the key
point for the CANON process. In period B (47—59 d),
oxygen was supplied with intermittent aeration and the
DO level was controlled within the range of 0.3—0.5
mg/L in accordance with the principles that appropriate
DO is beneficial to aerobic microorganism growth and
that the biofilm is exterior aerobic and interior anaerobic.
-
The concentration of NO2 -N decreased at the rate of 25%
every three days, which was beneficial for a subsequent
Anammox process. Under the condition, the removal rate
of NH4+-N was increased by above 80%, and the ammo-
nia nitrogen and total nitrogen concentrations in the ef-
fluent were below 5 and 15 mg/L, respectively.
the molar ratios of nitrate production to ammonium con-
sumption decreased gradually to 0.15, which was quali-
fied as the stoichiometric molar ratios of the CANON
process[5]. It was indicated that the CANON process had
been established in the reactor. Besides, the concentration
-
of NO3 -N increased slightly, because the excessive DO
in the influent changed nitrite into nitrate.
2.2 Performance of CANON process
In CANON process, there existed Anammox bacte-
ria and AOB in the reactor. Fig. 4 shows that the ranges
of FA and FNA were from 0.003 to 0.055 mg/L and from
0.000 2 to 0.002 5 mg/L, respectively. It has been ex-
perimentally demonstrated that AOB is insensitive to FA
and FNA, and the inhibition only takes place when FA
and FNA concentrations are higher than 10 mg/L and 0.2
mg/L, respectively[1,6]. FA concentration below 2 mg/L
and nitrite nitrogen below 35 mg/L have no inhibition on
Anammox bacteria[12]. As shown in Fig. 4, FA (FNA)
concentration was lower than 10 (0.2) mg/L when pH
was between 6.0 and 8.0, which indicated that FA and
FNA had little inhibition on the process of partial nitrifi-
cation and Anammox.

Fig.2 Profiles of nitrogen concentration in the reactor Fig.4 pH values of influent and effluent and profiles of
- NAR, FA, FNA under the condition of chemical
The stoichiometric ratios of NO3 -N production to equilibrium
+
NH4 -N consumption of Anammox and CANON were set
to be 0.26 and 0.13, respectively, as shown in Eqs. (2) 2.2.1 Effect of pH
and (3). From Fig. 3, it could be seen that the ratios With the alkalinity being consumed, the pH of solu-
were at the approach of baseline. By the end of period B, tion decreased, while it increased in the Anammox proc-
ess between NH4+-N and NO2 -N due to the hydrogen ion
-

consumption. Thus, the pH in effluent was lower than

that in influent and decreased with the decrease of pH in
influent, as shown in Fig. 4. The maximum removal rate
of NH4+-N was obtained at the optimal pH values be-
tween 7.6 and 7.9. The N-loading reached 480 mg/(L·d)
with the HRT of 1.25 h. The pH of the system has an ef-
fect on the partial nitrification and Anammox activity due
to its influence on the equilibrium of NH4+-N and NO2 -
-

Fig.3 Molar ratios of nitrate production to ammonium N. The optimal activity of nitrosomonas was obtained at
consumption and NLR the pH values between 7.9 and 8.2[1,13]. Moreover, recent

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 Transactions of Tianjin University Vol.19 No.4 2013
study shows that both Anammox bacteria and AOB have
a similar pH optimum of 8.0[14]. Experimental result was
in accordance with Ref. [14]. When pH was lower than
7.6 or higher than 7.9, the inhibition of partial nitrifica-
tion and Anammox would take place, and the nitrite ac-
cumulation increased rapidly. Fig. 4 shows that NAR
remains steady when pH is between 8.0 and 7.6. How-
ever, when pH is below 7.4, the nitrite concentration
starts to accumulate, indicating a steady inhibition of the
CANON process.
2.2.2 Effect of temperature
Both Anammox bacteria and AOB are sensitive to
temperature. The effect of temperature on CANON proc-
ess was studied at pH 7.9 and HRT 1.25 h, as shown in
Fig. 5. When the temperatures are 32, 30, 28, 26, 24, 22
and 20 ℃, the average removal rates of total nitrogen in
the system are 67%, 65%, 63%, 61%, 58%, 50% and 46%,
respectively. The removal rate of NH4+-N decreased, and
the effluent concentration of NH4+-N increased with the
decrease of temperature. Intuitively, the performance of
the CANON process was greatly limited by low tempera-
ture. Compared with the maximal N-removal rate of 67%
at 32 ℃, the maximal N-removal rate at 20 ℃ decreased
to 46%.
Fig.5 Nitrogen removal rate vs. temperature
The performance of the CANON process depends
on the competition among the involved organisms. On
one hand, nitrite oxidizing bacteria (NOB) compete with
AOB for oxygen, and on the other hand, they compete
with Anammox organisms for nitrite. Since the autotro-
phic organisms involved in the CANON process have
different growth rates and temperature coefficients, tem-
perature has different impacts on the growth rate of AOB,
NOB and Anammox bacteria. Thus, the change of tem-
perature can result in the change of process performance.
Ref. [15] indicated that it is most suitable for Anammox
bacteria to grow at 32 ℃. Ref. [14] showed that both
Anammox bacteria and AOB have a similar temperature
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optimum of 37 ℃ [12]. These mean that the activity of
Anammox organisms will decrease at a low temperature,
and that the activities of ammonium and nitrite oxidizing
bacteria will decrease at lower temperature. Above 25 ℃,
the maximum specific growth rate of AOB will be higher
than that of NOB under the conditions given by Ref.[16],
and the NOB can be out-competed effectively, whereas
the case is just opposite below 25 ℃. As shown in Fig.5,
it is beneficial to maintain the CANON process at 32 ℃.
However, for most wastewater treatment processes, it is
impractical to carry out denitrogenation at such a high
temperature.
2.3 Practical operation of wastewater from WWTP
With the successful reactor operation of synthetic
wastewater, the ammonium removal using secondary
effluent of municipal sewage will be investigated in the
same way.
Under the same operating condition, the concentra-
tion of DO was controlled within the range of 0.3 to 0.5
mg/L. The influent switched to fresh secondary effluent
with different dilution ratios on the 1st, 41st and 82nd
day. At each beginning, there was only ammonia nitrogen
without nitrite and nitrate nitrogen. Nitrite and nitrate
nitrogen occurred later in the secondary effluent, proba-
bly due to the presence of DO. With the increase of the
proportion of secondary effluent, the influent was com-
pletely secondary effluent of municipal sewage on the
88th day, and N-loading reached 360 mg NH4+-N/(L·d).
In the past 92 days, the ammonia nitrogen removal rate
was below 80% and the effluent concentration was above
5 mg/L. However, after the adoption of 6 d for the full
secondary effluent, the ammonia nitrogen and total nitro-
gen concentration in the effluent were 2 and 15 mg/L,
and their removal rates were 90% and 60%, respectively,
as shown in Fig. 6.
Fig.6 Concentrations of nitrogen and COD
 Peng Xinhong et al: CANON Process for Nitrogen Removal from Effluents of Municipal Sewage Treatment Plants

Besides ammonia nitrogen, there are also other or- 2475-2482.

ganics qualified by COD in the secondary effluent of ［6］ Kuypers M M M, Sliekers A O, Lavik G et al. Anaerobic
municipal sewage. From the 34th day, COD was moni- ammonium oxidation by Anammox bacteria in the Black
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sequencing batch biofilm reactor (SBBR) [J].
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"The reuse of urban recycling water: Water quality stan-
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［13］ Bernet N, Sanchez O, Cesbron D et al. Modeling and

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(Editor: Zhao Yang)

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