e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78

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A potential new process for improving nitrogen removal

in constructed wetlands—Promoting coexistence of
partial-nitrification and ANAMMOX

Zeqin Dong a,b,c,∗ , Tieheng Sun a

a Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences,
Shenyang 110016, People’s Republic of China
b Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
c Guizhou Institute of Environmental Science Research and Designing, Guiyang 550002, People’s Republic of China

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

Article history: Conventional nitrification by vertical flow constructed wetlands beds and denitrification by
Received 13 January 2006 horizontal flow constructed wetlands beds were considered as the main ways to remove
Received in revised form nitrogen in constructed wetlands. However, recent evidences showed that there are other
6 February 2007 microbial pathways for nitrogen removal which are based on partial-nitrification of ammo-
Accepted 15 April 2007 nium to nitrite with combining anaerobic ammonium oxidation (ANAMMOX). The full-scale
experiments, lasting 100 days in Evieu, east of France, showed that with appropriate design
and modification, for example dividing the depth of 80 cm conventional vertical flow beds
Keywords: into a 25 cm unsaturated layer and a 55 cm saturated layer, this new model cannot only
Nitrogen removal get a better performance in removal efficiency of total nitrogen, but also achieved a pro-
Partial-nitrification moted growth of ANAMMOX bacteria which will function to further remove the ammonium
ANAMMOX in horizontal flow beds. With fluorescent in situ hybridization (FISH) analysis, aerobic
Constructed wetlands design ammonia oxidizing bacteria (AOB) in conventional vertical flow CWs and ANAMMOX bac-
Fluorescence in situ hybridization teria in the interface between unsaturated–saturated layer of amelioration vertical flow
(FISH) analysis CWs and the followed horizontal flow CWs were tested. This paper described the design,
the performance and the results. Comparison experiments with conventional process of
nitrification–denitrification beds were also done at the same times; results indicated that
the two processes carried on are completely different pathways. Results of experiments,
nitrogen balance and FISH analysis proved that the process of partial-nitrification with
ANAMMOX can be realized in constructed wetlands and can obtain higher removal efficiency
of total nitrogen than conventional methods.
© 2007 Elsevier B.V. All rights reserved.

1. Introduction plant uptake and matrix adsorption. While nitrification–

denitrification is generally considered the main process for
Although mechanisms for nitrogen removal involved in nitrogen removal (Claudiane et al., 2006; Kadlec and Knight,
constructed wetlands (CWs) are manifold, which include 1996; Jan Vymazal, 2005; Tanita and Kimberly, 2006; Tanner
volatilization, ammonification, nitrification–denitrification, et al., 2002; Tjasa, 2006). Since 1990s, when an alternative

∗
Corresponding author at: Guizhou Institute of Environmental Science Research and Designing, Guiyang 550002, People’s Republic of
China. Tel.: +86 851 5520735; fax: +86 851 5520735.
E-mail addresses: dongfortune@hotmail.com, dongzheqin@163.com (Z. Dong).
0925-8574/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2007.04.009
70 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78
microbial pathway ANAMMOX for nitrogen removal was dis-
covered (Mulder et al., 1995; van de Graaf et al., 1995), several
ANAMMOX processes have been reported in rich-ammonium
wastewater treatment systems (Schmid et al., 2000a,b, 2003;
Egli et al., 2001; Helmer et al., 2001; Pynaert et al., 2003).
Recently the process has also been found in nature, such as
in marine sediments and anoxic water columns (Jetten et al.,
1999; Thamdrup and Dalsgaard, 2002; Dalsgaard et al., 2003;
Kuypers et al., 2003;). In treatment wetlands, ANAMMOX has
already been positively identified (Shipin et al., 2004). Ammo-
nia oxidizers have more versatile metabolism than previously
assumed, the aerobic nitrifier and anammox bacteria may be
natural partners in many oxygen-limited situations (Schmidt
et al., 2002). To investigate further and explore a feasible design
to develop these alternative pathways in constructed wetlands
win more concerns these days (Tanner et al., 2002; Kadlec et
al., 2005).
In order to meet increasingly stringent discharge stan-
dards, new control strategies for the removal of total nitrogen
from wastewater have to be developed in constructed wet-
lands. This paper reported firstly the feasible design for
Fig. 1 – (1) Full-scale experiments flow chart. (2) Sketch maps of construction of VFCWs (VF1-1). (3) Sketch maps of
construction of amelioration VF1-2.
promoting co-existence of partial-nitrification and ANAM-
MOX, and then provided evidences for developing new process
to remove total nitrogen efficiently in constructed wetlands.
2. Materials and methods
2.1. Study site
Full-scale experiments were carried out in the Evieu plant,
a constructed wetlands wastewater treatment plant built in
2003, in east France. This treatment plant serves for a small
community of about 220 people. It is made up of two-staged
vertical flow reed beds and one-staged horizontal flow reed
beds. These beds were all planted with Phragmites. The verti-
cal flow beds of the first stage, having a total area of 1.2 m2 /p.e.,
were divided into three identical alternately feeding units with
different design of depth or media. The vertical flow beds of
the second stage, 0.8 m2 /p.e., were divided into two identi-
cal alternately feeding units with different design of depth or
media. The horizontal flow beds are in two different config-
 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78 71

Table 1 – Design parameters of CWs reed beds

CWs reed beds Surface area (m2 ) Initial design Full-scale experiments design Porosity (%)
VF1-1 43.17 80 cm gravel (2–6 mm) + aeration pipe in layer of 40 cm 40
5–15 cm gravel (15–25 mm) + 15 cm scree (30–60 mm) (drain layer)

Amelioration VF1-2 28.07 80 cm gravel (2–6 mm) 25 cm gravel (2–6 mm) (unsaturated layer) 40
55 cm gravel (2–6 mm) (saturated layer)
5–15 cm gravel 5–15 cm gravel (15–25 mm) + 15 cm scree
(15–25 mm) + 15 cm scree (30–60 mm) (saturated drain layer)
(30–60 mm) (drain layer)
HF3-2 7.0 × 7.0 60 cm gravel (2–6 mm) 40

urations but with same depth; one is square in shape and
the other is rectangle, and they performed in parallel. The
initial purpose of the plant’s design was to enhance nitrifi-
cation with two-staged vertical flow beds, and denitrification
in horizontal flow beds according to conventional mecha-
nisms, oxidizing ammonium to nitrate and then reducing to
dinitrogen gas.
0.50 m. All the performance data of each bed and correspond-
ing weather conditions can be downloaded from the plant’s
datalogger. The design parameters of all experimental reed
beds are listed in Table 1.
2.3. Execution of the full-scale experiments

2.3.1. Cultivation conditions of ANAMMOX bacteria

2.2. Full-scale experiments design
Experiments lasted 100 days (from September to December,
2005). During the first 90 days, the main object is to promote
Since full nitrification needs more oxygen, nitrification in ver-
ANAMMOX bacteria in the aerobic–anoxic interface in ame-
tical flow reed beds would go much deeper or occupy much
lioration VF1-2. Amelioration VF1-2 functioned in the same
more area, so that nitrogen removal would be costly in CWs.
way as VF1-1, 3.5 days feeding with 7 days interval. The
One of the objectives of this study is to shorten the process
designed residence time in saturated layer of amelioration
and achieve a higher removal efficiency of total nitrogen.
VF1-2 was 1.18 days. For the experiments, the residence time
In the experiments, two initial vertical flow reed beds of the
was between 1.07 and 1.39 days, and the average residence
first stage were chosen as contrast. One is VF1-1 which has a
time was 1.15 (±0.13) days.
depth of 80 cm with aeration pipe, the other one VF1-2 has
Water temperature in saturated layer of amelioration VF1-
the same depth but without aeration pipe. The two beds have
2 was between 19.7 and 5.9 ◦ C, and its fluctuation according
the same media with porosity 40%. Before full-scale experi-
to the season can be seen in Table 2. The inlet pH of ame-
ments, the VF1-2 beds was modified into a new model with
lioration VF1-2 was between 7.82 and 8.26, and the average
a 25 cm unsaturated layer followed by a 55 cm saturated layer
value was 8.05 ± 0.16. Hydraulic loads of 24 h experiments of
at the bottom. Both of the two reed beds were connected to
these reed beds were between 0.484 and 0.706 m day−1 . Aver-
the same designed horizontal flow beds HF3-2. The flow chart
age hydraulic retention time of HF3-2 was 5 days. The inlet
of experiments is showed in Fig. 1(1), sketch maps of con-
wastewater parameters for full-scale experiments can be seen
struction of VFCWs and amelioration VF1-2 are showed in
in Table 3.
Fig. 1(2 and 3), respectively. Wastewater from Evieu commu-
nity entered into the first collection tank ( = 1.64 m). When the
water level reached 1.80 m, the pumps would function auto- 2.3.2. Pollutants sampling and analysis
matically to feed the vertical flow beds and make the water After the beds have functioned for 90 days, 10 days 24 h exper-
level drop from 1.80 to 1.10 m. VF1-1 and amelioration VF1- iments were carried out to evaluate the nitrogen removal effi-
2 were fed by pump1-1 and pump1-2, respectively, and each ciency. A 24 h composite samples were taken by model-3700R
feeding would last for 3.5 days, with an interval of 7 days. The refrigerated sequential sampler each hour from the inlet sta-
effluent was stored in the second collection tank ( = 1.31 m) tion, the second collection tank and from the outlet of HF3-2.
as a result of the hydraulic headloss. When the water level COD, SS, PO4 3− , KN, NH4 + , NO3 − , and NO2 − were anal-
arrived at 0.60 m, the pumps would work automatically to feed ysed by CEMAGREF-Lyon chemistry laboratory according to the
the HF3-2 beds and make the water level drop from 0.60 to French standard methods.

Table 2 – Water temperature (◦ C) changes in saturated layer of amelioration VF1-2

Experiment September October November December
periods

Feeding Rest Feeding Rest Feeding Rest Feeding Rest

Range 16–19.7 16–19.5 12.4–17.3 16–17.7 7.2–15.1 7.2–15.1 5.9–9.5 5.9–9.5

Mean 18.57 18.04 16.09 15.95 11.63 11.82 8.72 6.75
S.D. 0.22 0.37 0.52 0.54 1.37 1.04 0.64 0.41
72 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78

Table 3 – Full-scale 24 h experiments inlet wastewater qualities (mg L−1 )

COD SS TP PO4 3− KN NH4 + NO3 − NO2 − TN

Range 240–511 68–191 4.2–9.4 3.0–6.9 30.7–78.8 23.5–63.0 0.05–0.35 0.006–0.53 31.2–78.9
Mean 375.4 122.9 6.7 4.8 55.2 41.5 0.15 0.09 55.5
S.D. 80.7 36.1 1.6 1.4 14.7 12.8 0.10 0.15 14.8

2.3.3. Biomass sampling and bacteria detection

After the beds have functioned for 100 days, biomass of VF1-1
was taken from the media at the depth of −15 cm. Biomass
of amelioration VF1-2 was taken from the media at the depth
of −15 cm and the suspended water in saturated layer at the
depth between −25 and −40 cm by micro-pump. Biomass of
HF3-2 was also taken from the water at the depth from −10 to
−15 cm by micro-pump. For each reed bed, there should be 6
different sampling points, which are chosen according to their
distance from inlet point and aligned at 1, 2 and 3 m from inlet
in duplicate. All the biomass samples were prepared accord-
ing to standard method of fluorescence in situ hybridization
(FISH) analysis (Amann, 1995). Aerobic ammonia oxidizing
bacteria (AOB) and ANAMMOX bacteria in biomass samples
were detected by FISH analysis with Carl. Zeiss scanning Fig. 2 – Average removal efficiency (%) of pollutant in VF1-1
microscope and LSM image browser in CEMAGREF-Antony and amelioration VF1-2.
wastewater microbiology laboratory.
The 16S rRNA gene probes used in FISH analysis were:
Pla46 for cells of the order planctomycetales (Neef et al., 1998); were changed from aerobic (>200 MV), anoxic (200–200 MV) to
Amx820 specific for Candidatus ‘Brocadia anammoxidans’ and anaerobic (<−200 MV) and main reactions took place in less
Candidatus ‘kuenenia stuttgartiensis’ (Schmid et al., 2000a,b); oxygen environments. Denitrification in saturated layer con-
Amx0368 which targeted all anammox bacteria (Schmid et sumed COD and with partial-nitrification removed COD up
al., 2003); Eub338 for all bacteria (Daims et al., 1999); NEU653 to 71.96%.
for Nitrosomonas-like AOB bacteria (Juretschko et al., 1998; For SS, amelioration VF1-2 obtained higher removal effi-
Mobarry et al., 1996). Percent formamide in the hybridization ciency than VF1-1 did. This can be explained by their different
buffer and concentration of NaCl in the washing buffer were design and structure. In general, SS easy to be removed were
determined by the authors. The fluorophores were FITC for located within 15 cm on the top layer of each bed. Due to
AOB bacteria, Cy3 for Anammox bacteria and Cy5 for total saturated layer’s precipitation and adsorption of media, ame-
bacteria. lioration VF1-2 could accumulate more SS in the beds than
VF1-1.
For TP and PO4 -P removal, it was obvious that VF1-1 had
3. Results and discussions better performance than amelioration VF1-2, because that
phosphorus is mostly removed by adsorption. In VF1-1, phos-
3.1. Pollutants removal in VF1-1 and amelioration phorus was attached on media. While in amelioration VF1-2,
VF1-2 due to saturated layer, phosphorus was released at the outlet
again, where had an increased amount of phosphorus.
Ten days 24 h composite samples were analysed and COD, SS,
TP and PO4 -P average removal efficiency (%) were displayed in
Fig. 2. Results indicated that the performance of the two reed
beds had some certain differences.
For COD, its average removal efficiency in VF1-1 was
72.53%. This high removal efficiency accorded with the law
for full nitrification in VF1-1. In amelioration VF1-2, 71.96%
was also achieved, but this may be produced by the co-work
of partial-nitrification (nitrification) and denitrification. PH
and ORP measured at the interface of aerobic–anoxic and
outlet of amelioration VF1-2 can be seen in Figs. 3 and 4.
PH in the aerobic–anoxic interface staying mostly below
6.8 without decreasing, it showed that nitrification was
not full enough. The PH at the outlet increased to above
7.2, and generally the high PH was more suitable for the Fig. 3 – One function period PH of aerobic–anoxic interface
growth of ANAMMOX bacteria. Environments for the reaction and outlet of amelioration VF1-2.
 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78
Fig. 4 – One function period ORP of aerobic–anoxic interface
and outlet of amelioration VF1-2.
3.2. Nitrogen removal in VF1-1 and amelioration
VF1-2
KN, NH4 -N and TN were analysed and their average removal
efficiency can be seen in Fig. 5. In Fig. 6, the average removal
load (g m−3 day−1 ) of ammonium, nitrate and nitrite were
shown. Results indicated that more than half of ammo-
nium was removed in VF1-1, but amelioration VF1-2 whose
unsaturated layer was only 25 cm achieved 33.05% removal
efficiency of ammonium. Results also showed that KN was
positively removed as ammonium. With denitrification in
saturated layer, amelioration VF1-2 obtained higher total
nitrogen removal than that of VF1-1. Average nitrogen removal
load (g m−3 day−1 ) displayed in Fig. 6 attested that with con-
version of ammonium, nitrate were the main products in
VF1-1, which indicated the process in VF1-1 is conventional
nitrification. There was less increase of nitrate and nitrite in
amelioration VF1-2, which showed that not all the nitrate were
reduced in saturated layer by conventional denitrification. The
possible cause for the increase of nitrate was the reaction
ANAMMOX.
3.3. Nitrogen removal in HF3-2
Results of nitrogen removal analysis based on 24 h com-
posite samples displayed a completely different feature for
Fig. 5 – Average nitrogen removal efficiency (%) in VF1-1
and amelioration VF1-2.
73
Fig. 6 – Average nitrogen removal load (g m−3 day−1 ) in
VF1-1 and amelioration VF1-2.
ammonium removal. The ammonium amount removed in the
combination of VF1-2 and HF3-2 was two times of that in the
combination of VF1-1 and HF3-2. In both of the two processes,
total nitrogen removal achieved almost the same efficiency,
one was 54.83%, and the other was 53.94%. Results showed in
Fig. 7 indicated that ammonium removal was the dominating
one in the combination of VF1-2 and HF3-2 and it exerted on
total nitrogen removal.
The important evidences displayed in Fig. 8 indicated
that the two nitrogen removal pathways were completely
different from each other. Nitrate denitrification is the
predominating process in VF1-1 + HF3-2 while ammonium
oxidation is the major one in VF1-2 + HF3-2. That means
after most (or full) nitrification was obtained in VF1-1,
nitrogen removal would be undertaken according to con-
ventional nitrification–denitrification. In Fig. 8, nitrate and
nitrite were denitrified completely in VF1-1 + HF3-2. Also it
is found that in VF1-1 + HF3-2 a little part of ammonium
has been removed. It may be explained that due to partial-
nitrification in low-oxygen area, ammonium is converted
to nitrite then denitrified, when VF1-1 + HF3-2 is dominated
by heterotrophic bacteria while VF1-1 + HF3-2 by ANAMMOX
bacteria. Compared with partial-nitrification and ANAM-
MOX reaction, nitrification–denitrification was still the main
pathway for nitrogen removal in conventional vertical and
horizontal flow reed beds like VF1-1 + HF3-2.
It is worthy of paying more attention to the VF1-2 + HF3-2. In
this combination, ammonium oxidation in low-oxygen/anoxic
areas is the dominating combination in the removal of
total nitrogen, at the same time, there is also a part of
Fig. 7 – Average nitrogen removal efficiency (%) in HF3-2.
74 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78
Fig. 8 – Average nitrogen removal load (g m−3 day−1 ) in
HF3-2.
nitrite reduced and a small part of new nitrate produced
again. According to mechanisms of partial-nitrification and
ANAMMOX reaction, presumptions of reaction pathways
in amelioration VF1-2 followed by HF3-2 were deduced in
Figs. 9 and 10.
Experiments results of VF1-2 + HF3-2 were utilized to vali-
date presumptions of potential pathways for nitrogen removal
by ANAMMOX in HF3-2.
According to presumptions of Fig. 10, Eq. (3.1) (partial-
nitrification), Eq. (3.2) (ANAMMOX; Strous, 2000), Eq. (3.3) and
results displayed in Fig. 8, nitrogen balance is as follows.
NH4 + + 1.5O2 → NO2 − + 2H+ + H2 O
NH4 + + 1.32NO2 − + 0.066HCO3 − + 0.13H+
→ 1.02N2 + 0.26NO3 − + 0.066CH2 O0.5 N0.15 S0.05 + 2.03H2 O
NO3 − → NO2 −
Experiments results:
Fig. 9 – Presumptions of pathways for nitrogen removal in
amelioration VF1-2.
Fig. 10 – Presumptions of pathways for nitrogen removal in
VF1-2 + HF3-2.
total ammonium removal load: 2.76 g m−3 day−1 ;
final nitrite removal load: 0.178 g m−3 day−1 ;
final nitrate production load: 0.08 g m−3 day−1 .
Nitrogen is balanced as presumptions:
Eq. (3.1)
1.478 g m−3 day−1 NH4 + −→ 3.778 g m−3 day−1 NO2 − ;
1.12 g m−3 day−1 NH4 + + 3.778 g m−3 day−1 NO2 −
Eq. (3.2)
−→ 1.00 g m−3 day−1 NO3 − ;
Eq. (3.1)+Eq. (3.2)
2.598 g m−3 day−1 NH4 − −→ removal;
− Eq. (3.3)
(3.1) 1.00 g m−3 day−1 NO3 −→ 0.744 g m−3 day−1 NO2 − ;
−0.178 g m−3 day−1 NO2 − → N2 ;
0.168 g m−3 day−1 NH4 + + 0.566 g m−3 day−1 NO2 −
Eq. (3.2)
−→ 0.150 g m−3 day−1 NO3 − ;
Eq. (3.1)+Eq. (3.2)+Eq. (3.3)
2.766 g m−3 day−1 NH4 + −→ removal.
From nitrogen balance above, 2.766 g m−3 day−1 ammo-
(3.2) nium should be removed theoretically according to presump-
tions, and there were 276 g m−3 day−1 ammonium removed
in fact. Experiments better accorded with presumptions
(3.3) for ammonium removal by partial-nitrification and ANAM-
MOX in HF3-2 with amelioration VF1-2 as pretreatment
process. But for production of nitrate, there should be
0.150 g m−3 day−1 NO3 − in HF3-2, actually there were only
0.08 g m−3 day−1 nitrate produced in HF3-2. This part of nitrate
may be the required limit for maintaining the growth of
ANAMMOX bacteria. The other part of nitrate, which has
Fig. 11 – Average removal efficiency (%) of pollutant in
HF3-2.
 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78 75

disappeared, has a big possibility of being utilized by micro-
bials or assimilated by plants of reeds. Deduction above
also indicated that ammonium should be removed by co-
work of partial-nitrification and ANAMMOX in this kind of
design CWs.
3.4. Pollutants removal in HF3-2
A 24 h composite samples of the process VF1-1 + HF3-2 and
VF1-2 + HF3-2 were taken from the second collection tank and
at the outlet of HF3-2. The average removal efficiencies of COD,
SS and PO4 -P were shown in Fig. 11.
With HF3-2, the removal efficiencies of COD and SS for the
combination of VF1-1 + HF3-2 were 67.01% and 81.01%. But
for the amelioration VF1-2 + HF3-2, higher removal efficien-
cies have been achieved, for example, COD removal rate at
75.15% and SS at 85.89%. It is obvious that COD from VF1-1 is
mainly removed by the consumption of denitrification in HF3-
2. But there were no more information available for explaining
the higher removal efficiency of COD for the combination
of amelioration VF1-2 + HF3-2 till now. There were no more
nitrates from amelioration VF1-2 needing to be denitrified in
HF3-2, and its removal efficiency was higher than that from
VF1-1.
From results showed in Figs. 7 and 8, possible presumption
for the higher COD removal efficiency was that heterotrophic
bacteria may be coexisting with ANAMMOX bacteria, in that
condition, COD is needed to be carbon sources for nitrite
reduction. May be in horizontal beds, existed some differ-
ent districts such as low-oxygen areas and anaerobic areas,

Fig. 12 – (1) Amelioration VF1-2 (−25 to −40 cm) ANAMMOX bacteria cells appear purple. At right separated pictures: top left,
Amx820 probe (Cy3); top right, EUB probe (Cy5); bottom left, phase contrast; bottom right, overlapping picture. (2) HF3-2 (−10
to −15 cm) ANAMMOX bacteria cells appear purple. (3b) VF1-1 (−15 cm) AOB bacteria cells appear shamrock, because
overlap of FITC (NEU653 probe-green) and Cy5 (EUB probe-blue) labeling. At right separated pictures: top left, NEU 653 probe;
top center, Amx probe; top right, EUB probe. (3a) Amelioration VF1-2 (−15 cm) AOB bacteria cells appear shamrock.
76 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78
Fig. 12 – (Continued ).
where series different reactions by aerobic-anammox bac-
teria or anaerobic bacteria would take place. At first, in
low-oxygen areas, ANAMMOX bacteria could oxide ammo-
nium into nitrite which would be used as electron acceptor
to reduce ammonium into dinitrogen gas; meanwhile, pro-
portional nitrate was produced provided sufficient COD for
this part of nitrate’s reduction, subsequently nitrate would
be reduced into nitrite which can play the role of electron
acceptor again for reaction of ANAMMOX. Then reaction
would go on in anaerobic environments, heterotrophic bacte-
ria may consume COD again to reduce part of nitrate which
was produced by the last step of ANAMMOX reaction. If
these presumptions were logical, that means with propor-
tional sufficient COD, not only coexistence of heterotrophic
bacteria and ANAMMOX bacteria can be promoted in hori-
zontal CWs, but also total nitrogen and COD removal can get
improved.
For phosphorus removal, for the combination of amelio-
ration VF1-2 and HF3-2, the removal efficiency was 44.09%,
but for VF1-1 and HF3-2, it was only 19.34%. More information
is needed for making clear if there were other mechanisms,
accompanied by ANAMMOX bacteria reaction in horizontal
reed beds for phosphorus removal except adsorption.
3.5. AOB and ANAMMOX bacteria
With FISH analysis, AOB bacteria were detected in the biomass
samples of VF1-1 and amelioration VF1-2 at depth −15 cm,
but no AOB bacteria was detected in suspended biomass sam-
ples of amelioration VF1-2. ANAMMOX bacteria were tested
in suspended biomass samples of amelioration VF1-2 and fol-
lowed HF3-2. Results can be seen in Fig. 12(1–3). Only the probe
Amx820 gives positive results on the different samples. On
the outlet sample, positive ANAMMOX bacteria appeared as
dispersed cells whereas in the biomass sample, the positive
ANAMMOX bacteria appeared as small aggregate.
Results of FISH analysis attested that there were ANAM-
MOX bacteria existing in the interface of unsaturated-
saturated layer of amelioration VF1-2 and in the low-oxygen
layer of HF3-2. Results also showed that ANAMMOX bacteria
seem to be able to develop at the present of AOB bacteria.
4. Conclusions
Full-scale studies have provided evidences that promoting
coexistence of partial-nitrification and ANAMMOX can be
 e c o l o g i c a l e n g i n e e r i n g 3 1 ( 2 0 0 7 ) 69–78
realized in CWs with feasible design, which will improve
total nitrogen removal efficiency. However, several key points
should be mentioned.
1. CWs was suitable reactor for storing biomass and promot-
ing growth of ANAMMOX; it is very important to design and
build aerobic–anoxic interfaces in CWs which play big role
in increasing biomass of ANAMMOX bacteria.
2. Even water temperature in CWs was 19.7∼5.9 ◦ C, ANAM-
MOX reactions still worked; nitrogen balance and FISH
analysis accorded with presumptions of co-work of partial-
nitrification and ANAMMOX.
3. Controlling PH in pretreatment CWs can help to promote
growth of ANAMMOX bacteria; and proportional sufficient
ammonium was necessary for ANAMMOX bacteria to com-
pete with heterotrophic bacteria in horizontal reed beds.
4. ANAMMOX bacteria can coexist with heterotrophic bacte-
ria. With appropriation design, heterotrophic bacteria can
consume oxygen and produce a low-oxygen environment
for ANAMMOX and reduce nitrate to nitrite to promote
reaction of ANAMMOX.
5. Existence of proportional COD can promote coexistence
of heterotrophic bacteria and ANAMMOX bacteria. All
these bacteria can utilize nitrite and thereby the nitrogen
removal can be improved.
CWs are very complicated systems, where nitrogen
removal is easily influenced by multi-factors such as design
parameters of CWs construction, hydraulic loads and wastew-
ater qualities at the inlet, operation and weather conditions,
etc. Experiments results revealed that partial-nitrification
and ANAMMOX can be realized in CWs, which provide a
potential new alternate process for improving total nitrogen
removal. Still more information is needed to be investigated
and explored in CWs, such as the species of ANAMMOX bac-
teria, effect of phosphorus, optimal design parameters and so
on. This is the primary report of study, further more studies on
optimizing ANAMMOX process in CWs are still going on with
full-scale experiments.
Acknowledgements
Evieu CWs wastewater treatment plant provided the site for
the full-scale study, the chemistry laboratory of CEMAGREF-
Lyon and the microbiology laboratory of CEMAGREF-Antony
conducted professional analysis. Grateful thanks goes to all of
them for their generous helps.
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