Ecological Engineering 81 (2015) 266271

Contents lists available at ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Nitrogen removal pathways in a tidal ow constructed wetland under

ooded time constraints
Luzhen Li a,b , Chunguang He b, * , Guodong Ji a, * , Wei Zhi a , Lianxi Sheng b
a

Key Laboratory of Water Sediment Sciences, Ministry of Education, Department of Environmental Engineering, Peking University, Beijing 100871, China
State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School Environment, Northeast Normal University,
Changchun 130117, China
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 16 September 2014
Received in revised form 17 March 2015
Accepted 9 April 2015
Available online xxx

The present study explored treatment performance and nitrogen removal mechanisms of a single-stage
tidal ow constructed wetland (TFCW) under ooded time ranging from 12 to 48 h. The TFCW achieved
high and stable COD (7794%), NH4+N (5582%), and TN (6084%) removal efciencies simultaneously,
without costly aeration. Quantitative response relationships between nitrogen transformation rates and
nitrogen functional genes were established, and these relationships conrmed that, under the FT
constraints, the anammox 16S rRNA was the key factor of the NH4+N transformation rates, whereas,
(napA + narG) was the key factor regulating the NO3
N transformation rate. The analysis also revealed
that the simultaneous nitrication, anammox, and denitrication (SNAD) process was the dominant
nitrogen removal pathway in the TFCW.
2015 Elsevier B.V. All rights reserved.

Keywords:
Tidal ow constructed wetland (TFCW)
Flooded time (FT)
Nitrogen functional genes
Anammox

1. Introduction
Tidal ow constructed wetlands (TFCWs) are one of the most
signicant developments in constructed wetlands (CWs) in the last
decade, having substantially improved the treatment capacity of
organic matter and ammonia nitrogen by overcoming the limited
oxygen supply of traditional CWs (Sun et al., 2005; Wu et al.,
2011a). However, the total nitrogen (TN) removal efciency in
TFCWs is not ideal because of the high oxygen content (Ju et al.,
2014; Cui et al., 2012).
The tidal ow principle, which includes four operational
procedures (ll-contact-drain-rest), distinguishes TFCWs from
traditional CWs (Sun et al., 2006). The theory behind the process
of nitrogen removal in TFCWs is as follows. First, ammonium
cations (NH4+N) are adsorbed on matrix, pores, and surfaces
when wetland cells are ooded. Second, as wetland cells drain,
matrix, pores immediately ll with air, according to Ficks law,
which shows that oxygen transfer is favored over nitrication of
adsorbed NH4+N. Finally, in the next ood cycle, nitrate (NO3
N)
and nitrite (NO2
N) desorb into bulk water, where they are
denitried into atmospheric nitrogen (Austin, 2006; Chang et al.,
2014). Therefore, the ooded time (FT) of TFCWs plays a key role in
forming effective anoxic conditions that are favorable for reducing
oxidized-N (NO2
N/NO3
N) to improve removal total nitrogen
(TN).

* Corresponding authors. Tel.: +86 431 89165612; fax: +86 431 89165611.
http://dx.doi.org/10.1016/j.ecoleng.2015.04.073
0925-8574/ 2015 Elsevier B.V. All rights reserved.

The process of nitrogen removal in CWs is extremely complex

and includes biological (i.e., ammonication, nitrication, denitrication, plant uptake, biomass assimilation, and dissimilatory
nitrate reduction) and physico-chemical routes (i.e., ammonia
volatilization, and adsorption) (Saeed and Sun, 2012), but the main
nitrogen removal process is the microbial metabolic pathway, with
removal of around 8996% of the nitrogen (Lin et al., 2002).
Removal occurs via several metabolic pathways, including
conventional or partial nitricationdenitrication and anaerobic
ammonium oxidation (anammox). Management of environmental
variables will improve the nitrogen removal efciency and lead to
one preferential nitrogen removal pathway (Wu et al., 2011b; Ge
et al., 2014; Rajagopal and Bline, 2011).
The microbial metabolic pathways in TFCWs are more
complicated than in conventional CWs because of multiple
periodical ood/drain or oxic/anoxic cycles. The FT of TFCWs
may inuence hydraulic retention time (HRT), with greater
nutrient removal being observed with larger FTs (Sakadevan and
Bavor, 1999). An increase in HRT also causes a decrease in oxygen
uptake rate (Muda et al., 2011), and the FT affects microorganisms
within TFCWs by changing the oxygen transfer rate. To date,
information on major microbial removal nitrogen pathways in
TFCWs remains scarce.
In the present study, tidal operations were used in CWs with
different FTs. Nitrogen functional genes (amoA,nxrA, napA, narG,
nirS, qnorB, nosZ, and anammox 16S rRNA) were quantied by realtime quantitative polymerase chain reaction (real-time PCR).
Microbial consortia responsible for nitrogen removal in relation to

L. Li et al. / Ecological Engineering 81 (2015) 266271

changes in FTs were investigated under FT constraints. To acquire

more complete insight into the role of microorganisms, the
relationship between nitrogen transformation rates and nitrogen
functional genes was evaluated.
2. Materials and methods
2.1. TFCW set-up and operation
The TFCW consisted of an inuent tank, metering pump, CW
bed, and efuent tank. The CW bed (length  width  height = 40
cm  20 cm  120 cm), with an effective working volume of 8.8 L,
was divided into a water distribution layer (020 cm), treatment
layer (20100 cm), and water collection layer (100120 cm). The
treatment layer was lled with volcanic rocks (particle diameter:
810 mm) and the water collection layer was lled with gravel
(diameter: 1020 mm).
Operation of the CW followed the tidal ow principle
(ll-contact-drain-rest). Operation was controlled by a metering
pump (ProMinent1, China) and a programmable timer. Wastewater from the holding tank was pumped into the CW within 10 min
via batch mode. After the tank was lled, the bed was kept
saturated for a certain time period, designated as the FT. Thereafter,
all of the wastewater was drained rapidly (10 min) into the efuent
tank and the bed was left unsaturated with water for 12 h. The
experiment began on 5 January 2012 and was divided into six
stages over the 188 days experimental period: (1) start-up Stage
(FT = 12 h), from 5 January 2012 to 17 January 2012; (2) Stage A
(FT = 12 h), from 18 January 2012 to 21 February 2012; (3) Stage B
(FT = 18 h), from 22 February 2012 to 28 March 2012; (4) Stage C
(FT = 24 h), from 29 March 2012 to 3 May 2012; (5) Stage D
(FT = 36 h), from 4 May 2012 to 7 June 2012; and (6) Stage E
(FT = 48 h), from 8 June 2012 to 12 July 2012. The system was kept in
an indoor area. Environmental temperature ranged from 15.1 to
27.0  C during the experiments.
2.2. Sample collection and determination
Water samples were collected from the inlet and outlet at least
three times during each phase and were analyzed immediately
after collection at the Key Laboratory of Water and Sediment
Sciences at Peking University. To minimize variability in the
experiment, synthetic wastewater containing ammonium, COD,
and phosphate concentrations was used in this study. Inuent
articial wastewater was prepared using C6H12O6, NH4CL, and
KH2PO4 dissolved in Beijing groundwater (4.95.9 mg/L of NO3

N). The synthetic wastewater was prepared daily in a feeding tank
and pumped into the CW though umes in the distribution layer.
COD was determined with a HACH DR2800 (HACH, USA). The three
nitrogenous compounds were measured using a UV-1800 spectrophotometer (SHIMADZU, Japan). All variables were analyzed
according to standard analytical procedures (Ji et al., 2012). The

267

inuent wastewater characteristics of each phase are summarized

in Table 1.
Microorganism samples were collected from preburied columns at the end of the week in each phase. During each sampling
event, the preburied column was removed from the CW bed,
placed in an ice incubator, and immediately sent to the Laboratory
for DNA extraction. Soil DNA kits D5625-01 (Omega, USA) were
used to extract and purify the total genomic DNA from the samples.
Extracted genomic DNA was detected by 1% agarose gel
electrophoresis and stored at
20  C.
2.3. Quantication of the nitrogen functional genes
Abundance of bacterial gene 16S rRNA (abbreviated as bacterial
16S rRNA) and nitrogen functional genes, including amoA, nxrA,
napA, narG, nirS, qnorB, nosZ, and anammox bacteria gene 16S rRNA
(abbreviated as anammox 16S rRNA), were determined by
real-time PCR using a MyiQ2 Real-Time PCR Detection System
(Bio-Rad, USA) with the uorescent dye SYBR-Green approach.
Amplication was performed in 20 mL reaction mixtures, including
10 mL of SYBR Green I PCR master mix (Applied Biosystem, USA),
1 mL of template DNA (sample DNA or plasmid DNA for standard
curves), 0.5 mL of forward primers, 0.5 mL of reverse primers, and
8 mL of sterile water. Each real-time PCR amplication consisted of
a total of 40 cycles. The primer sequences and real-time PCR
protocol followed Ji et al. (2012).
2.4. Statistical analysis
One-way analysis of variance (ANOVA) was conducted to assess
the statistical signicance of differences among the various
microorganism groups. Relationships between groups were
determined by Pearsons correlation coefcient (r). Stepwise
regression models were constructed to determine the quantitative
response relationships between nitrogen transformation rates and
the functional genes through the use of SPSS Statistics 18.0
(StatSoft, USA). In all tests, signicant effects were those with pvalues < 0.05.
3. Results
3.1. Effect of FTs on pollution reduction
The removal efciency of pollutants in the TFCWs varied greatly
with different FTs. The removal efciency of chemical oxygen
demand (COD) ranged from 77% to 94%, with an inow
concentration of 200 mg/L (Fig. 1a). Inow concentrations of
NH4+N and TN were approximately 35 and 40 mg/L, respectively.
As FT increased from 12 to 48 h, removal efciencies of NH4+N and
TN increased from 55% to 82% and from 60% to 84%, respectively
(Fig. 1b and c). The average removed concentrations increased from
19.30 to 26.31 mg/L for NH4+N and from 24.44 to 31.06 mg/L for
TN, respectively. The efuent concentrations of NO2
N and NO3

Table 1
Inuent wastewater characteristics in each phase (average  SD) (mg/L).

COD
NH4+N
NO2
N
NO3
N
TN
pH
DO

Phase 1 (12 h)
2148 days

Phase 2 (18 h)
5583 days

Phase 3 (24 h)
90118 days

Phase 4 (36 h)
125153 days

Phase 5 (48 h)
160188 days

199.9  8.16
32.80  1.35
0.0083  0.0049
5.37  1.7
38.18  2.75
7.43  0.31
8.68  0.17

240.93  11.93
34.20  2.52
0.0057  0.0021
5.9  0.24
39.79  2.45
7.38  0.46
8.37  0.16

207.6  16.96
34.57  3.6
0.0040  0.0036
5.00  0.14
39.57  4.9
7.36  0.10
8.17  0.28

206.1  5.27
37.50  1.73
0.0087  0.0067
4.47  0.27
41.98  1.55
7.34  0.02
7.44  0.37

204.07  16.51
34.80  0.62
0.0097  0.0058
4.74  0.33
39.55  0.74
7.32  0.03
7.02  0.34

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L. Li et al. / Ecological Engineering 81 (2015) 266271

Fig. 3. Absolute abundance of nitrogen transformation functional genes.

was higher than the accumulation rate with other FTs, the
accumulation rate still remained under 0.05 g/m3/day (Fig. 2).
3.2. Effect of FT on the absolute abundance of nitrogen functional
genes

Fig. 1. Concentrations and removal efciency of COD (a), NH4+N (b), TN (c) and
effuent concentration of NO3
N, NO2
N, and DO (d).

N were both under 0.6 mg/L (Fig. 1d). The efuent concentrations
of DO decreased slightly as FTs increased from 12 to 48 h (Fig. 1d).
FT played an important role in the nitrogen transformation
rates. The transformation rates of TN, NH4+N, and NO3
N
decreased sharply as FT increased. NH4+N and TN removal rates
nearly halved when FT increased from 12 to 48 h. Under this
scenario, the NO3
N removal rate decreased from 5.69 to 1.70 g/
m3/day. Although the NO2
N accumulation rate with a CT of 18 h

Fig. 2. Transformation rates of TN, NH4+N, NO3
N, and NO2
N.

As FT increased, the absolute abundance value of different

nitrogen functional genes changed signicantly (Fig. 3). Anammox
16S rRNA is often regarded as the marker of anammox that
converts NH4+N and NO2
N to N2 (Tsushima et al., 2007). The
absolute abundance of anammox 16S rRNA increased from
1.57  106 to 2.70  106 copies/g as FT increased from 12 to 48 h,
indicating a positive correlation with FT (r = 0.763, p = 0.13).
Therefore, an increase in FT was benecial to potential anaerobic
ammonium oxidation in our TFCW.
The amoA and nxrA genes are often regarded as the marker of
oxidizing NH4+N to NO2
N (Dionisi et al., 2002) and oxidizing
NO2
N to NO3
N (Poly et al., 2008), respectively. Both genes are
involved in nitrication. Ammonia oxidation, which involves
amoA, is thought to be the rate-limiting step for nitrication in
most systems (Kowalchuk and Stephen, 2001). When FT increased
from 12 to 48 h, the absolute abundance of amoA decreased from
1.75  105 to 2.14  104 copies/g, and the absolute abundance of
nxrA decreased from 2.29  104 to 5.74  103 copies/g. Thus, both
genes showed a negative correlation with FT (r =
0.803 and

0.608, p = 0.10 and 0.28, respectively). Increasing FT was
disadvantageous to potential nitrication in the TFCW.
Membrane-bound nitrate reductase (Nar) and periplasmic
nitrate reductase (Nap) are two different types of dissimilative
nitrate reductase that may be found in the same or different
bacteria (Deiglmayr et al., 2004). Nar/Nap expression is dominant
under oxic/anoxic conditions. Nap is encoded by napA, which is
often regarded as the marker of conversion of NO3
N into NO2

N in the aerobic. Nar is encoded by narG, which is often regarded as
the marker of conversion of NO3
N into NO2
N in the anaerobic.
As both napA and narG genes are involved in the rst step of
denitrication, we could use the absolute abundance of (napA +
narG) to represent the potential of denitrication in the step of
converting NO3
N into NO2
N (Lpez-Gutirrez et al., 2004; Bru
et al., 2007). The absolute abundance of napA showed a negative
correlation with FT (r =
0.946, p = 0.02) and decreased from
2.95  106 to 3.56  105 copies/g, and the absolute abundance of
narG showed a positive correlation with FT (r = 0.667, p = 0.22) and
increased from 4.67  105 to 1.40  106 copies/g, as FT increased
from 12 to 48 h.
As the key enzyme in the dissimilatory denitrication process,
nitrite reductase is catalyzed by the products of two different
nitrite reductase genes: the copper-containing product of nirK and

L. Li et al. / Ecological Engineering 81 (2015) 266271

269

the cytochrome cd1-containing product of nirS (Braker et al., 1998).

The nirK and nirS genes, which are involved in the second
denitrication step, are often regarded as the markers of
converting NO2
N into NO (Yan et al., 2003). The absolute
abundance of nirK was too low to be quantied by real-time PCR in
our TFCW. The absolute abundance of nirS was positively
correlated with FT (r = 0.808, p = 0.10), with a minimum value of
5.43  102 copies/g during a FT of 24 h and a maximum value of
3.79  103 copies/g during a FT of 48 h.
The qnorB gene is often regarded as the marker of converting NO
into N2O, which is involved in the third denitrication step (Braker
and Tiedje, 2003). The absolute abundance of qnorB was positively
correlated with FT (r = 0.505, p = 0.39), with a minimum value of
1.22  104 copies/g during a FT of 18 h and a maximum value of
4.59  104 copies/g during a FT of 36 h. The nosZ gene is often
regarded as the marker of converting N2O into N2, which is
involved in the last denitrication step (Scala and Kerkhof, 1998).
The absolute abundance of nosZ was positively correlated with FT
(r = 0.727, p = 0.16), with a minimum value of 8.02  103 copies/g
during a FT of 24 h and a maximum value of 4.22  104 copies/g
during a FT of 48 h.
4. Discussion
TFCW could increase the transportation rates of the oxgen,
which could further improve the removal efciency of COD and
NH4+N, but this process could decrease the removal efciency of
TN. Previous studies have demonstrated that the concept model
of nitrogen conversion in TFCWs as follows: NH4+N is adsorbed
onto the wetland medium in the ooded time and is converted to
NO2
N/NO3
N in the drained time; the residual oxidized-N
(NO2
N + NO3
N) is released and converted by anammox or
denitrication in the ooded time of next cycle, which means that
the longer FT will improve the deoxidation of oxidized-N. In this
study, the minimum FT of 12 h was greater than in previous
studies (the maximum FT of previous study was 8 h) and the
removal efciency of TN was 84%, even higher than the NH4+N.
Furthermore, the efuent concentrations of NO2
N and NO3
N
were both under 0.6 mg/L in the whole running process, which
means that the deoxidation of NO3
N was complete. Therefore,
the control and balance of FT is more important in the application
of TFCW.
In order to identify key functional genes that determine
nitrogen transformation process rates in the TFCW, a series of
stepwise regression models were built to provide a linear
quantitative measure of functional genes association with nitrogen
transformation rates. Seven functional genes, (i.e., anammox 16S
rRNA, (napA + narG), amoA, nxrA, nirS, qnorB, and nosZ) were used as
candidate variables in stepwise regression analysis to associate
with nitrogen transformation rates. Results showed three NH4+N,
NO3
N, and TN equations were successfully established (Table 2).
Under the FT constraint, our results indicated that the anammox
16S rRNA and (napA + narG) genes were the rate-limiting factors
that solely determined the NH4+N and NO3
N transformation
rates, respectively. For example, the NH4+N transformation rate

Fig. 4. Nitrogen transformation pathways with functional genes in TFCWs (a) and
transformation limiting pathways of NH4+N (b), NO3
N (c) and NO2
N (d).

was estimated to be 1.8-fold smaller when the absolute abundance

of anammox 16S rRNA increased from 1.57  106 to 2.70  106
copies/g. The NO3
N transformation rate decreased 3.4 times
when the absolute abundance of (napA + narG) increased from
3.42  106 to 1.75  106 copies/g.
While the rate-limiting gene of nitrogen transformation rate
can be derived from stepwise regression analysis that uses the
absolute abundance of single functional genes as input variables,
utilization of functional gene groups (ratio or summation of
different functional genes) may provide additional explanatory
information that is benecial in characterizing the dynamics of
nitrogen transformation processes (Zhi and Ji, 2014; Wang et al.,
2015). All regression equations were successfully established (also
in Table 2). Fig. 4a shows the nitrogen transformation pathways
with functional genes in TFCWs.
The signicant variables that explained the largest part of the
variation in NH4+N transformation rate were the values of
anammox 16S rRNA/(napA + narG) (R2 = 0.868, p < 0.050) and
amoA/anammox 16S rRNA (R2 = 0.990, p < 0.010). The anammox
16S rRNA/(napA + narG) denoted NO3
N accumulation as the
anammox 16S rRNA produced NO3
N, while the napA + narG
consumed NO3
N. Zhou et al. (2014) found that the anammox
activity decreased when the rst denitrication step (NO3

N ! NO2
N by napA/narG) was inhibited in the sediment of an
inland river. This may explain that the NO3
N accumulation
hinders
anammox
(NH4+N + NO2
N ! NO3
N + N2)
by

Table 2
Quantitative response relationships between nitrogen transformation rates and functional genes abundance (n = 5).
Stepwise regression models

Improved stepwise regression models

Equations

R2

p-Value

Equations

R2

p-Value

NH4+N =
6.45  10
6 anammox 16S rRNA + 28.05

0.819

0.035

0.990

0.010

NO3
N = 2.09  10
6 (napA + narG)
1.58

NO2
N

0.915
N.A.

0.011
N.A.

NH4+N = 21.53 anammox 16S rRNA/(napA + narG)
150.89

amoA/anammox 16S rRNA + 44.01
NO3
N = 3.83n  rA/qnorB + 24.53 amoA/(napA + narG) + 0.63
NO2
N = 0.006amoA/nxrA
807.33 amoA/bacterial 16S rRNA + 0.01

0.996
0.998

0.004
0.002

N.A. = not available.

270

L. Li et al. / Ecological Engineering 81 (2015) 266271

anammox 16S rRNA, which was the rate-limiting factor of NH4+N

to declined NH4+N transformation. The amoA/anammox 16S
rRNA, denoted as NO2
N accumulation, showed a positive
relationship with NH4+N transformation, which is not unexpected
given that both amoA and anammox 16S rRNA were primarily
involved in NH4+N conversion. Fig. 4b shows the dynamics of
NH4+N transformation processes. The process coupling between
partial nitrication (NH4+N ! NO2
N by nxrA) and anammox
(NH4+N + NO2
N ! NO3
N + N2 by anammox 16S rRNA) has
been named CANON (completely autotrophic nitrogen removal
over nitrite) (Dijkman and Strous, 1999). Dalsgaard demonstrated
that anammox was coupled to denitrication in the anoxic water
column. Thus, the main NH4+N removal pathway was the
coupling of the CANON with the NO3
N ! NO2
N by napA/
narG (Dalsgaard et al., 2003).
The signicant variables that explained the largest part of the
variation in the NO3
N transformation rate were the values of nxrA/
qnorB (R2 = 0.903, p < 0.050) and amoA/(napA + narG) (R2 = 0.996,
p < 0.005). The nxrA/qnorB denoted NO3
N accumulation, as the
nxrA gene was the main process contributing to NO3
N production,
while the qnorB gene consumed NO3
N for the NO3
N content
would have been correlated signicantly with the measured N2O
emission (Giles et al., 2012; Angnes et al., 2013). The amoA/
(napA + narG) denote NO2
N accumulation, as both amoA and
narG/napA genes were primarily involved in NO2
N production.
Therefore, NO2
N accumulation was positively related with NO3

N transformation rate. The more NO2
N accumulated, the more
NO3
N transformed. Fig. 4c shows that the main pathway for
NO3
N removal was denitrication. Although anammox could
remove NO3
N when NO3
N was converted to NO2
N by narG/
napA, some research has also demonstrated that aerobic ammonium oxidation, rather than nitrate reduction, provides a direct local
source of nitrite for anammox in the suboxic zone (Lam et al., 2007;
Wang et al., 2012).
The signicant variables explaining the largest part of the
variation in NO2
N accumulation rate were the values of amoA/
nxrA (R2 = 0.817, p < 0.050) and amoA/bacterial 16S rRNA(R2 = 0.998,
p < 0.005). The amoA/nxrA and amoA/bacterial 16S rRNA both
denote NO2
N accumulation, as the amoA produced NO2
N,
while the nxrA consumed NO2
N. Fig. 4d shows the dynamics of
NO2
N accumulation processes. Zhang et al. (2011) showed that
AOB (ammonia oxidation bacteria) were more competitive than
NOB (nitrite oxidizing bacteria), leading to an accumulation of
NO2
N. The efuent concentration of NO2
N in the FT of 18 h
was highest, as the ratio amoA/nxrA in the FT of 18 h was more than
twice that of other FTs. Therefore, the main NO2
N accumulation
pathway was the coupling of the rst with the second nitrication
step.
In conclusion, both ammonia-oxidizing and denitrifying microorganisms are involved in, and contribute to, nitrogen transformation, in addition to the simultaneous nitrication, anammox, and
denitrication (SNAD) process, all of which were identied at the
molecular level in our TFCW, under FT constraints. The co-existence
of the SNAD processes may assist in the simultaneous removal of
nitrogen and organic carbon in a single system, in accordance with
high COD and nitrogen removal efciency (especially the TN removal
efciency) in our TFCW. This means that it is possible to improve the
removal efciency of COD and nitrogen at the same time in TFCWs if
the FT is controlled for the process of SNAD.
5. Conclusions
A single-bed TFCW was constructed and operated under
variable ooded time (FT) ranging from 12 to 48 h. The TFCW
simultaneously achieved high and stable COD (7794%), NH4+N
(5582%), and TN (6084%) removal efciency. Quantitative

responses suggested that, under the FT constraints, the anammox

16S rRNA was the key factor of the NH4+N transformation rates,
whereas, (napA + narG) was the key factor regulating the NO3
N
transformation rate. Finally, the simultaneous nitrication, anammox, and denitrication (SNAD) process was the dominant
nitrogen removal pathway in the TFCW.
Acknowledgments
The National Natural Science Foundation of China (no.
51179001), the National Key Technology R&D Program of China
(no. 2012BAJ25B01), China National Special Funds of Science and
Technology for Control and Remediation of Water Pollution
(no. 2012ZX07201-001), and the Collaborative Innovation Center
for Regional Environmental Quality provided support for this
study.

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