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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.
∗
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- promoting co-existence of partial-nitrification and ANAM-
covered (Mulder et al., 1995; van de Graaf et al., 1995), several MOX, and then provided evidences for developing new process
ANAMMOX processes have been reported in rich-ammonium to remove total nitrogen efficiently in constructed wetlands.
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 2. Materials and methods
in marine sediments and anoxic water columns (Jetten et al.,
1999; Thamdrup and Dalsgaard, 2002; Dalsgaard et al., 2003; 2.1. Study site
Kuypers et al., 2003;). In treatment wetlands, ANAMMOX has
already been positively identified (Shipin et al., 2004). Ammo- Full-scale experiments were carried out in the Evieu plant,
nia oxidizers have more versatile metabolism than previously a constructed wetlands wastewater treatment plant built in
assumed, the aerobic nitrifier and anammox bacteria may be 2003, in east France. This treatment plant serves for a small
natural partners in many oxygen-limited situations (Schmidt community of about 220 people. It is made up of two-staged
et al., 2002). To investigate further and explore a feasible design vertical flow reed beds and one-staged horizontal flow reed
to develop these alternative pathways in constructed wetlands beds. These beds were all planted with Phragmites. The verti-
win more concerns these days (Tanner et al., 2002; Kadlec et cal flow beds of the first stage, having a total area of 1.2 m2 /p.e.,
al., 2005). were divided into three identical alternately feeding units with
In order to meet increasingly stringent discharge stan- different design of depth or media. The vertical flow beds of
dards, new control strategies for the removal of total nitrogen the second stage, 0.8 m2 /p.e., were divided into two identi-
from wastewater have to be developed in constructed wet- cal alternately feeding units with different design of depth or
lands. This paper reported firstly the feasible design for media. The horizontal flow beds are in two different config-
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.
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
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 0.50 m. All the performance data of each bed and correspond-
the other is rectangle, and they performed in parallel. The ing weather conditions can be downloaded from the plant’s
initial purpose of the plant’s design was to enhance nitrifi- datalogger. The design parameters of all experimental reed
cation with two-staged vertical flow beds, and denitrification beds are listed in Table 1.
in horizontal flow beds according to conventional mecha-
nisms, oxidizing ammonium to nitrate and then reducing to
2.3. Execution of the full-scale experiments
dinitrogen gas.
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
Fig. 9 – Presumptions of pathways for nitrogen removal in Fig. 11 – Average removal efficiency (%) of pollutant in
amelioration VF1-2. 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- cies have been achieved, for example, COD removal rate at
bials or assimilated by plants of reeds. Deduction above 75.15% and SS at 85.89%. It is obvious that COD from VF1-1 is
also indicated that ammonium should be removed by co- mainly removed by the consumption of denitrification in HF3-
work of partial-nitrification and ANAMMOX in this kind of 2. But there were no more information available for explaining
design CWs. the higher removal efficiency of COD for the combination
of amelioration VF1-2 + HF3-2 till now. There were no more
3.4. Pollutants removal in HF3-2 nitrates from amelioration VF1-2 needing to be denitrified in
HF3-2, and its removal efficiency was higher than that from
A 24 h composite samples of the process VF1-1 + HF3-2 and VF1-1.
VF1-2 + HF3-2 were taken from the second collection tank and From results showed in Figs. 7 and 8, possible presumption
at the outlet of HF3-2. The average removal efficiencies of COD, for the higher COD removal efficiency was that heterotrophic
SS and PO4 -P were shown in Fig. 11. bacteria may be coexisting with ANAMMOX bacteria, in that
With HF3-2, the removal efficiencies of COD and SS for the condition, COD is needed to be carbon sources for nitrite
combination of VF1-1 + HF3-2 were 67.01% and 81.01%. But reduction. May be in horizontal beds, existed some differ-
for the amelioration VF1-2 + HF3-2, higher removal efficien- 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- 3.5. AOB and ANAMMOX bacteria
teria or anaerobic bacteria would take place. At first, in
low-oxygen areas, ANAMMOX bacteria could oxide ammo- With FISH analysis, AOB bacteria were detected in the biomass
nium into nitrite which would be used as electron acceptor samples of VF1-1 and amelioration VF1-2 at depth −15 cm,
to reduce ammonium into dinitrogen gas; meanwhile, pro- but no AOB bacteria was detected in suspended biomass sam-
portional nitrate was produced provided sufficient COD for ples of amelioration VF1-2. ANAMMOX bacteria were tested
this part of nitrate’s reduction, subsequently nitrate would in suspended biomass samples of amelioration VF1-2 and fol-
be reduced into nitrite which can play the role of electron lowed HF3-2. Results can be seen in Fig. 12(1–3). Only the probe
acceptor again for reaction of ANAMMOX. Then reaction Amx820 gives positive results on the different samples. On
would go on in anaerobic environments, heterotrophic bacte- the outlet sample, positive ANAMMOX bacteria appeared as
ria may consume COD again to reduce part of nitrate which dispersed cells whereas in the biomass sample, the positive
was produced by the last step of ANAMMOX reaction. If ANAMMOX bacteria appeared as small aggregate.
these presumptions were logical, that means with propor- Results of FISH analysis attested that there were ANAM-
tional sufficient COD, not only coexistence of heterotrophic MOX bacteria existing in the interface of unsaturated-
bacteria and ANAMMOX bacteria can be promoted in hori- saturated layer of amelioration VF1-2 and in the low-oxygen
zontal CWs, but also total nitrogen and COD removal can get layer of HF3-2. Results also showed that ANAMMOX bacteria
improved. seem to be able to develop at the present of AOB bacteria.
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 4. Conclusions
is needed for making clear if there were other mechanisms,
accompanied by ANAMMOX bacteria reaction in horizontal Full-scale studies have provided evidences that promoting
reed beds for phosphorus removal except adsorption. 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 77
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