Nitrogen removal and mass balance in newly-formed Myriophyllum aquaticum mesocosm during a single 28-day incubation with swine wastewater treatment

1. Research article
Nitrogen removal and mass balance in newly-formed Myriophyllum
aquaticum mesocosm during a single 28-day incubation with swine
wastewater treatment
Feng Liu a, b, *, 1
, Shunan Zhang a, c, 1
, Yi Wang a, b
, Yong Li a, b
, Runlin Xiao a, b
,
Hongfang Li a, c
, Yang He a, c
, Miaomiao Zhang a, b
, Di Wang a, c
, Xi Li a, b
, Jinshui Wu a, b, **
a
Key Laboratory of Agro-ecological Processes in Subtropical Regions, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan, 410125, PR
China
b
Changsha Research Station for Agricultural & Environmental Monitoring, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan,
410125, PR China
c
Graduate University of Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e i n f o
Article history:
Received 7 March 2015
Received in revised form
10 October 2015
Accepted 9 November 2015
Available online 19 November 2015
Keywords:
Myriophyllum aquaticum
Swine wastewater
Nitrogen removal
Plant uptake
Nitrification and denitrification
Nitrogen mass balance analysis
a b s t r a c t
The aim of this research was to assess the applicability of Myriophyllum (M.) aquaticum for swine
wastewater treatment. Nitrogen (N) removal processes were investigated in M. aquaticum mesocosms
with swine wastewater (SW), 50% diluted swine wastewater (50% SW), and two strengths of synthetic
wastewater, 200 mg NH4
þ
À N LÀ1
(200 NH4
þ
À N) and 400 mg NH4
þ
À N LÀ1
(400 NH4
þ
À N). During a
28-day incubation period, the average NH4
þ
À N and TN removal rates were 99.8% and 94.2% for 50% SW
and 99.8% and 93.8% for SW, which were greater than 86.5% and 83.7% for 200 NH4
þ
À N, and 73.7% and
74.1% for 400 NH4
þ
À N, respectively. A maximum areal total nitrogen (TN) removal rate of
157.8 mg N mÀ2
dÀ1
was found in M. aquaticum mesocosms with SW. During the incubation period, the
observed dynamics of NO3
À
À N concentrations in water and gene copy numbers of ammonia-oxidizing
archaea (AOA), ammonia-oxidizing bacteria (AOB), nirK and nirS in soil unraveled strong nitrification and
denitrification processes occurring in M. aquaticum mesocosms with swine wastewater. The N mass
balance analysis indicated that plant uptake and soil N accumulation accounted for 17.9e42.2% and 18.0
e43.8% of the initial TN load, respectively. The coupled nitrification and denitrification process was
calculated to account for, on average, 36.8% and 62.8% of TN removal for 50% SW and SW, respectively.
These findings demonstrated that the N uptake by M. aquaticum contributed to a considerable proportion
of N removal. In particular, the activities of ammonia-oxidizing and denitrification microbes responsible
for nitrification and denitrification processes in M. aquaticum mesocosm accelerated NH4
þ
À N and TN
removal from swine wastewater.
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Wastewater from animal operations that contain high concen-
trations of nutrients, chemical oxygen demand (COD) and
suspended particles have caused the degradation of water quality
in many rivers and lakes (Schaafsma et al., 2000; Xu and Shen,
2011; Li et al., 2014). Low-cost constructed wetlands (CWs) have
been utilized to control pollution from animal wastewater in many
sites all over the world (Cronk, 1996; Vymazal, 2011). Surface-flow
CWs with macrophytes have shown good performance in treating a
variety of wastewater (Vymazal, 2013a). In addition, two types of
surface-flow CWs of continuous-marsh and marsh-pond-marsh
have been preferred as useful methods for swine wastewater
treatment in the U.S. (Poach et al., 2004).
Macrophytes are a key biological component and play an
important role in removing N in CWs. Plant growth assimilates
* Corresponding author. Changsha Research Station for Agricultural & Environ-
mental Monitoring, Institute of Subtropical Agriculture, Chinese Academy of Sci-
ences, Hunan, 410125, PR China.
** Corresponding author. Changsha Research Station for Agricultural & Environ-
mental Monitoring, Institute of Subtropical Agriculture, Chinese Academy of Sci-
ences, Hunan, 410125, PR China.
E-mail addresses: liufeng@isa.ac.cn (F. Liu), jswu@isa.ac.cn (J. Wu).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Journal of Environmental Management
journal homepage: www.elsevier.com/locate/jenvman
http://dx.doi.org/10.1016/j.jenvman.2015.11.020
0301-4797/© 2015 Elsevier Ltd. All rights reserved.
Journal of Environmental Management 166 (2016) 596e604

2. nutrients to form organic compounds such that when the plants are
harvested, the N and P are removed from the polluted water bodies
(Vymazal, 2007, 2011). Plant vegetation provides a carbon source
and a high surface area for the improvement of microbial activities
in wetlands (Cronk, 1996). Radial oxygen loss (ROL) creates
oxygenation areas adjacent to plant roots, which stimulates aerobic
processes such as nitrification (Brix, 1997; Stottmeister et al., 2003).
The accumulation of decaying plant litter within the bottom layer
of wetlands, which is always anoxic, supports enough bioavailable
carbon sources for the denitrification process to occur (Vymazal,
2013b). Hence, plant vegetation effectively increases the microbi-
al transformation of N, thus enhancing N removal (Tanner et al.,
1995; Maltais-Landry et al., 2009). Coupled nitrification and deni-
trification in wetlands contribute to N removal from ammonia-rich
animal wastewater (Morgan and Martin, 2008). Ammonia-
oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA)
are two groups of prokaryotes, which are responsible for a rate-
limiting step (the oxidation of ammonia to nitrite) in nitrification
(Hou et al., 2013). Analysis of the functional genes of nitrate
reductase (narG), nitrite reductase (nirK and nirS), and nitrous oxide
reductase (nosZ) can provide valuable information on the diversity
of denitrifiers in environment samples (Kandeler et al., 2006).
The N removal abilities of CWs depend on plant species,
wastewater type, and N loading rates. Plant uptake contributes to N
removal rates of 3e47% (Gottschall et al., 2007; Vymazal, 2007). A
thorough understanding of the role of plants in CWs can provide
important information for the optimization of wetland design. For
animal wastewater treatment, the selected plant species should be
tolerant of high ammonium concentrations and have high pro-
ductivity to enable rapid nutrient uptake (Cronk, 1996). Myr-
iophyllum aquaticum is a widespread submergent and emergent
herb that can grow with stem branches of up to 1 m and that readily
grows in tropical regions of the world (Torres Robles et al., 2011).
Despite its invasive status in natural water bodies, the risk of
M. aquaticum spreading to other ecosystems is manageable in CWs.
M. aquaticum can be planted in a drainage ditch to mitigate agri-
cultural non-point runoff pollution. Study results show that the
vigorous growth of M. aquaticum contributes to the rapid accu-
mulation of organic carbon in ditch soils, which enhances the soil
phosphorus adsorption capacity (Liu et al., 2013). On other way, it is
reported that M. aquaticum roots have the ability to create oxidized
areas and release organic compounds at the rootesediment inter-
face that play important roles in plant metal accumulation
(Teuchies et al., 2012). Overall, there is limited available research
regarding the use of M. aquaticum in improving environmental
quality. Meanwhile, the potential of M. aquaticum for animal
wastewater treatment have been unknown.
To understand the potential of M. aquaticum for N removal from
wastewater, the main objectives of this study were: (1) to deter-
mine the changes in NH4
þ
À N, NO3
À
À N, and TN concentrations
in M. aquaticum mesocosms; (2) to investigate the effects of
wastewater types with two strengths on the dynamics of the
abundance of ammonia-oxidizing and denitrifying functional
genes; and (3) to evaluate N removal pathways in M. aquaticum
mesocosms through a N mass balance analysis.
2. Materials and methods
2.1. The study plant, wastewater and soil
Field observations showed that M. aquaticum can grow well in
drainage ditches, streams, ponds, and wetlands in subtropical
China from March to December when the daily mean temperature
is above 10 C (Liu et al., 2013). M. aquaticum used in this study was
transplanted from a novel constructed drainage ditch. Swine
wastewater for the study was collected from an anaerobic lagoon in
a pig-breeding farm, and the soil was sampled from a paddy field,
both of which were located within the Changsha Research Station
for Agricultural Environmental Monitoring of the Chinese
Academy of Sciences in Hunan Province, P.R. China. The sampled
swine wastewater contained 416.8 mg LÀ1
NH4
þ
À N, 0.41 mg LÀ1
NO3
À
À N, 458.1 mg LÀ1
total nitrogen (TN), 31.6 mg LÀ1
total
phosphorus (TP), and 1142.5 mg LÀ1
COD. The soil properties were
as follows: TN of 1.86 g kgÀ1
, TP of 0.83 g kgÀ1
, total carbon content
of 21.93 g kgÀ1
, a pH value of 6.71 (measured at soil to water so-
lution ratio of 1:2.5 w/v), and a loam texture consisting of 32.6%
sand, 41.1% silt, and 26.3% clay.
2.2. Setup of mesocosms
The experimental mesocosms were investigated in a green-
house from August 14th to September 11th. During the experi-
mental incubation period, air temperatures in the greenhouse were
between 18.8 C and 37.4 C.
Twelve square plastic tanks with dimensions of 50 cm
length  40 cm width  50 cm depth were used to prepare
M. aquaticum mesocosms. Ten kilograms of air-dried paddy soil was
added to each tank to produce a soil layer with approximately 5 cm
in depth, which were firstly incubated with tap water for 3 days.
One hundred plant shoots with a uniform length of 20 cm and a
total fresh weight of approximately 90 g were planted into the soil
layer of each tank, after which they were incubated with tap water
for another 5 days. After the added tap water was completely dis-
charged, M. aquaticum mesocosm was used as a batch treatment
system with the addition of 15 L of wastewater at one time. To
investigate the differences in N removal from different types of
wastewater in the M. aquaticum mesocosm, two types of waste-
water, each with two strengths, were prepared. The sampled swine
wastewater from an anaerobic lagoon was used as high-strength
swine wastewater (SW). By diluting SW with tap water at a 1:1
(v/v) ratio, the 50% diluted swine wastewater (50% SW) was pre-
pared as low-strength swine wastewater. Based on the NH4
þ
À N
concentrations in the two different strengths of swine wastewater,
two strengths of synthetic wastewater e 200 mg LÀ1
NH4
þ
À N(200
NH4
þ
À N) and 400 mg LÀ1
NH4
þ
À N solution (400 NH4
þ
À N)d
were prepared with ammonium sulfate. Each wastewater was
spiked into M. aquaticum mesocosms in triplicates. According to
high N removal rates of 98% for NH4
þ
À N and 97% for TN obtained
in a field-scale integrated M. aquaticum wetlands with a hydraulic
retention time of about one month (Li et al., 2015), an experimental
time of 28 days was used as the incubation period in this study.
During the whole experimental period, distilled water was added
to replace evaporation losses and maintain a constant water depth
of 7.5 cm.
2.3. Sampling and analysis
After the study wastewater was added into M. aquaticum mes-
ocosms, 100 mL water samples were collected by a 50 mL syringe
on days 0, 1, 3, 7, 10, 14, 17, 22, 25 and 28. The concentrations of
NH4
þ
À N and NO3
À
À N in the wastewater were measured using a
fully automated flow-injection system (FIA-star 5000 analyzer, Foss
Tecator, H€ogan€as, Sweden). To analyze TN, water samples were first
digested using K2S2O8eNaOH solution, and the digested NO3
À
À N
was measured using the automated flow-injection system. Addi-
tionally, the water quality parameters of pH, DO, and temperature
(T) were measured using a portable multiple parameter meter
(SG68-ELK, Mettler Toledo, Switzerland) at a water depth of 5 cm in
M. aquaticum mesocosms at 09:00 AM.
By using a cylindrical stainless auger with a diameter of 2 cm,
F. Liu et al. / Journal of Environmental Management 166 (2016) 596e604 597

3. five soil samples were collected from the M. aquaticum mesocosms
on days 0, 1, 7, 14, and 28; the samples were homogenized to create
a combined soil sample with a wet weight of approximately 100 g.
The fresh soils were extracted using a 2 M KCl solution, and the
extracted soil NH4
þ
À N, NO3
À
À N, and TN contents were
measured using the method as described in the published paper (Fu
et al., 2012). Soil water content was measured using the oven-
drying method at 105 C for 24 h. All results of soil analyses pre-
sented in this study were presented on an oven-dried basis.
Microbial total DNA was extracted from approximately 0.5 g of
fresh soil using aUltraClean™ Soil DNA Isolation Kit (MoBio, USA).
The primer pairs of AOB amoA 1F and amoA 2R (Rotthauwe et al.,
1997), AOA Arch-amoA 23F and Arch-amoA 616R (Sahan and
Muyzer, 2008), nirK 517F and 1055R (Henry et al., 2004), and nirS
263F and 950R (Throb€ack et al., 2004) were employed for quanti-
fying bacterial amoA, archaea amoA, nirK and nirS, respectively. The
qPCR assays were performed with a sequence detection system
(ABI prism 7900, Applied Biosystem, USA). The reaction was per-
formed in a volume of 50 mL reaction mixture with 25 mL PCR mix
(Tiangen, China), 0.2 mmol LÀ1
forward and reverse primer, and
50 ng of template DNA. Thermal cycling conditions for bacterial and
archaea amoA gene were as follows: 95 C for 2 min; 40 cycles of
95 C for 15 s, 55 C for 30 s, and 72 C for 30 s. The thermal cycling
program for nirK and nirS were as follows: 94 C for 2 min, 35 cycles
of 94 C for 30 s, 51 C for 1 min, and 72 C for 1 min. Standard
curves were obtained from serial 10-fold dilutions of plasmids
containing bacterial amoA, archaea amoA, nirK, and nirS gene,
respectively.
On day 28, M. aquaticum was cut at the water level and collected.
Plant samples were dried at 105 C for 30 min and oven dried at
80 C until constant weight was reached. The dried plant material
was ground, passed through a 1 mm sieve, and stored at 4 C until
use. The TN content in M. aquaticum was determined after digestion
with H2O2 and HClO4.
2.4. Statistical analysis
The N removal rate (NRR, %) was calculated using the following
equation:
NRR ¼
Ci À Cf
Ci
 100 (1)
where Ci and Cf are the initial (on day 0) and final (on day 28) N
concentrations in wastewater, respectively.
According to Gebremariam and Beutel (2008), areal NH4
þ
À N
and TN removal rates were calculated as the difference in N loss
over the interval days (2e4 d) divided by the area of the mesocosms
(0.2 m2
) and expressed in units of mg per square meter per day (mg
mÀ2
dÀ1
). Especially, the dissipations of NH4
þ
À N and TN were fast
in M. aquaticum mesocosms, and small differences of NH4
þ
À N and
TN concentrations between 14 days and 28 days led to very low
areal NH4
þ
À N and TN removal rates in the latter experiment
period. Thus, only the experimental data observed over days 1e14
were used to calculate areal NH4
þ
À N and TN removal in this
study.
The N mass balance analysis was based on the calculation that
the initial TN load in the M. aquaticum mesocosms was equal to the
sum of the final N load in water, the TN uptake by plant, the TN
accumulation in soil, the ammonia volatilization, and the estimated
N loss from the nitrification and denitrification processes. The
calculation method was expressed as Eq. (2), which was similar to
that described in a published paper (Peng et al., 2012). Eq. (3) was
used to estimate the amount of N removal by nitrification and
denitrification.
Qi ¼ Qf þ Qpu þ Qsa þ Qv þ Qen (2)
Qen ¼ Qi À Qf À Qpu À Qsa À Qv (3)
where Qi and Qf are initial and final N load in the wastewater in
M. aquaticum mesocosms, which are respectively calculated by
multiplying Ci and V, and Cf and V (V ¼ 15 L), Ci and Cf represent the
same variables as in Eq. (1); Qpu is TN uptake by plant, Qsa is TN
accumulation in soil, Qv is the ammonia volatilization and is
neglected in this study because pH values in the range of 4.47e7.94
were observed for the mesocosms with swine and synthetic
wastewater, and Qen is the estimated TN removal in the nitrification
and denitrification processes.
The TN uptake by plant was calculated by multiplying the dry-
weight biomass by the TN content in plant. TN accumulation in
soil was calculated as the difference between the soil TN contents
on day 0 and 28 multiplied by the soil weight in mesocosms. The
comparison of N concentrations in the two strengths of swine and
synthetic wastewater was performed using a paired-sample t-test
to evaluate the N removal efficiency during the incubation period.
The differences in areal NH4
þ
À N and TN removal rates, soil N
contents, and plant N uptake among M. aquaticum mesocosms with
different wastewaters were performed using the Duncan's new
multiple-range test of one-way ANOVA. The abundance of AOB,
AOA, nirK, and nirS in M. aquaticum mesocosms on days 0, 7, 14, and
28 were compared by the Duncan's new multiple-range test too. At
a p value equal to or less than 0.05, the difference was considered
statistically significant. All analyses were performed using SPSS v.
13.0.
3. Results
3.1. Physicochemical parameter variation in wastewater
The values of pH, dissolved oxygen (DO), and temperature in
wastewater are presented in Table 1. Two strengths of swine
wastewater had pH values that varied between 6.67 and 7.94.
Average pH values of 7.30 for 50% SW and 7.28 for SW were nearly
neutral. The pH values of synthetic wastewater decreased with
increasing incubation time, from 6.48 for 200 NH4
þ
À N and 6.28
for 400 NH4
þ
À N on day 1e4.47 and 4.50, respectively, on day 28,
and average pH values of 5.42 for 200 NH4
þ
À N and 5.21 for 400
NH4
þ
À N were acidic. The lowest DO values for 50% SW and SW
were 3.94 mg LÀ1
and 1.92 mg LÀ1
, respectively, each measured on
Table 1
Statistics of pH, dissolved oxygen (DO), and temperature (T).
Parameters Wastewater
50% SW SW 200 NH4
þ
À N 400 NH4
þ
À N
pH
Mean ± SD (n) 7.30 ± 0.12 7.28 ± 0.17 5.42 ± 0.30 5.21 ± 0.24
MineMax 6.91e7.85 6.67e7.94 4.47e6.48 4.50e6.28
DO (mg LÀ1
)
Mean ± SD (n) 4.92 ± 0.32 4.41 ± 0.56 6.35 ± 0.26 6.36 ± 0.29
MineMax 3.94e6.60 1.92e6.35 5.51e7.65 5.30e7.55
Water T (
C)
Mean ± SD (n) 28.2 ± 1.56 28.1 ± 1.59 28.1 ± 1.54 28.3 ± 1.53
MineMax 20.4e32.7 20.2e32.9 20.3e32.8 20.5e32.3
SD: standard deviations (n ¼ 24). Different wastewater used in this study are
abbreviated as follows: diluted 50% swine wastewater (50% SW), swine wastewater
from an anaerobic lagoon (SW), 200 mg LÀ1
NH4
þ
À N solution (200 NH4
þ
À N), and
400 mg LÀ1
NH4
þ
À N solution (400 NH4
þ
À N). These abbreviations in Tables 2 and
3 and Figs. 1e4 share the same meaning.
F. Liu et al. / Journal of Environmental Management 166 (2016) 596e604598

4. day 1. DO levels in swine wastewater exhibited a general increasing
trend during the incubation period. DO concentrations of both
strengths of synthetic wastewater were similar, with an average
value of approximately 6.35 mg LÀ1
. Average DO concentrations of
two strengths swine wastewater were significantly lower than
those of synthetic wastewater (p 0.01). Throughout the experi-
mental period, the same average water temperature of 28.2 C was
observed in all wastewater.
3.2. Nitrogen removal from wastewater
The changes in NH4
þ
À N concentration over the 28-day incu-
bation period indicated that NH4
þ
À N removal efficiencies in
swine wastewater were higher than those in synthetic wastewater
(Fig. 1 (a)). NH4
þ
À N removal rates of 97% were observed for both
50% SW and SW on day 14, but the rates were 74% for 200 NH4
þ
À
N and 64% for 400 NH4
þ
À N. The smallest NH4
þ
À N removal rate
of 73.7% was observed for 400 NH4
þ
À N on day 28. The NO3
À
À N
concentrations in two strengths swine wastewater showed great
variation, increasing from 1 mg LÀ1
on day 1 to peak concentra-
tions of 20.8 mg LÀ1
for 50% SW and 61.9 mg LÀ1
for SW on day 10
(Fig. 1 (b)). However, the NO3
À
À N concentrations in two strengths
synthetic wastewater remained at a relatively low level of
1.1 mg LÀ1
throughout the incubation period. Two strengths swine
wastewater had higher TN removal efficiencies than two strengths
synthetic wastewater (Fig. 1 (c)). The removal rates of TN were
approximately 94% for both 50% SW and SW, 83.7% for 200 NH4
þ
À
N and 74.1% for 400 NH4
þ
À N on day 28.
Areal NH4
þ
À N and TN removal rates decreased as N loading
rates decreased during the experimental period of 1e14 days. The
areal NH4
þ
À N removal rates ranged from 4.64 to
86.9 mg N mÀ2
dÀ1
were observed for 50% SW, which were
significantly higher than those for 200 NH4
þ
À N during 1e3 and
4e6 days, and 7.45e169.5 mg N mÀ2
dÀ1
for SW, significantly
higher than those for 400 NH4
þ
À N during 1e3, 4e6, and 7e10
days (Fig. 2a). SW had significantly higher areal TN removal rates
compared with 50% SW, 200 NH4
þ
À N, and 400 NH4
þ
À N treat-
ments during 1e3 and 4e6 days (Fig. 2b). The areal NH4
þ
À N and
TN removal rates were, on average, 6.7% and 16.7% higher in
M. aquaticum mesocosms with 50% SW than those with 200
NH4
þ
À N, and they were 76.9% and 39.9% higher in M. aquaticum
mesocosms with SW than those with 400 NH4
þ
À N.
3.3. Soil nitrogen accumulation
Soil NH4
þ
À N, NO3
À
À N and TN contents of M. aquaticum
mesocosms are shown in Fig. 3. Soil NH4
þ
À N contents of
M. aquaticum mesocosms with swine wastewater generally
decreased with increasing incubation time. M. aquaticum meso-
cosms with synthetic wastewater had more soil NH4
þ
À N content
than those with swine wastewater. The maximum soil NH4
þ
À N
content of 96.7 mg kgÀ1
dry soil was observed in M. aquaticum
mesocosms with 400 NH4
þ
À N, which was significantly greater
than in those with 50% SW and SW (p 0.01) (Fig. 3 (a)).
M. aquaticum mesocosms with SW had the maximum soil NO3
À
À
N content of 49.5 mg kgÀ1
dry soil on day 14. In general, the dif-
ference in soil NO3
À
À N contents of M. aquaticum mesocosms with
swine and synthetic wastewater was negligible by day 28 (Fig. 3
(b)). Soil TN contents were enhanced in M. aquaticum mesocosms
with the higher strength wastewater. The difference in soil TN
contents between M. aquaticum mesocosms with SW and 400
NH4
þ
À N was significant (p 0.05) (Fig. 3 (c)). Soil TN accumula-
tion accounted for 16.6e43.8% of the initial TN load (Table 2).
3.4. Plant nitrogen uptake
The harvested biomass and TN content of M. aquaticum of the
mesocosms with swine wastewater and synthetic wastewater were
shown in Fig. 4. The mesocosm with 400 NH4
þ
À N produced the
maximum plant dry matter yield of 45.0 g (equaling 2250 kg haÀ1
),
Fig. 1. Changes in NH4
þ
À N (a), NO3
À
À N (b) and TN (c) concentrations in wastewater
during the experimental incubation period. The error bars represent the standard
deviations (n ¼ 3). The error bars in Figs. 2e4 share the same meaning.
F. Liu et al. / Journal of Environmental Management 166 (2016) 596e604 599

5. and the mesocosm with SW had the minimum of 37.8 g (equaling
1890 kg haÀ1
). The TN content of M. aquaticum ranged from 29.4 to
32.5 g kgÀ1
dry weight, and the highest and lowest TN contents
were observed in the mesocosms with 400 NH4
þ
À N and SW,
respectively. The amount of N uptake by M. aquaticum ranged from
1.06 to 1.44 g (equaling 53e72 kg haÀ1
) for the mesocosms with
different wastewater. The N uptake by M. aquaticum presented the
lowest percentage (15.9%) of the initial TN load in the mesocosms
with SW and the highest (46.2%) in the mesocosms with 200
NH4
þ
À N (Table 2).
3.5. Abundance of AOB, AOA, nirK and nirS
Except on day 0, the differences in abundance of AOB, AOA, nirK
and nirS were observed between M. aquaticum mesocosms with SW
and 400 NH4
þ
À N on days 7, 14 and 28 (Table 3). The abundance of
AOB and AOA ranged from 2.03 Â 107
to 4.56 Â 108
and from
1.51 Â 107
to 3.78 Â 108
gene copies gÀ1
dry soil, respectively.
M. aquaticum mesocosms with SW had significantly higher AOB
gene copy numbers than that with 400 NH4
þ
À N (p 0.05).
Abundance of AOA in M. aquaticum mesocosms with SW was also
significantly higher than that with 400 NH4
þ
À N on days 7 and 14,
but significantly less on day 28 (p 0.05). The ranges of
4.12 Â 109e8.98 Â 109 gene copies g-1 dry soils for nirK and
1.66 Â 107e1.61 Â 108 for nirS were determined in M. aquaticum
mesocosms with SW, and the ranges of 2.32 Â 109e4.61 Â 109 for
nirK and 7.61 Â 106e4.65 Â 107 for nirS in M. aquaticum
mesocosms with 400 NH4
þ
À N, respectively. The gene copy
numbers of nirS in M. aquaticum mesocosms with SW were
significantly higher than those with 400 NH4
þ
À N on days 7 and
28 (p 0.05).
4. Discussion
4.1. Nitrogen removal efficiencies in M. aquaticum mesocosms
Macrophytes in wetlands play important roles in N removal
Fig. 2. Areal-based NH4
þ
À N (a) and TN (b) removal in M. aquaticum mesocosms with
swine and synthetic wastewater.
Fig. 3. The accumulation of NH4
þ
À N (a), NO3
À
À N (b) and TN (c) in soil. The
different letters in the graph indicate a significant difference at p 0.05. The letters in
Fig. 4 have the same meaning.
F. Liu et al. / Journal of Environmental Management 166 (2016) 596e604600

6. (Cronk, 1996; Li et al., 2013). Comparisons of plant species in N
removal suggest that proper species selection is important for the
design and planning of wetland management (Saeed and Sun,
2012). The present study focused on the potential of
M. aquaticum for N removal from NH4
þ
À N-dominated wastewater
in experimental mesocosms. The changes in NH4
þ
À N, NO3
À
À N
and TN concentrations over the 28-day incubation period indicated
that wastewater strengths affected the N removal efficiency in
M. aquaticum mesocosms. The NH4
þ
À N and TN concentrations
decreased more rapidly to near zero value in low-strength
wastewater than in high-strength wastewater (Fig. 1 (a, c)). This
phenomenon was consistent with the changes in N concentrations
in three strengths of dairy wastewater treated by ecological treat-
ment systems (Morgan and Martin, 2008).
Despite their lower NH4
þ
À N and TN removal rates,
M. aquaticum mesocosms with high-strength wastewater showed
higher areal N removal rates than those with low-strength waste-
water (Fig. 2). The maximum areal NH4
þ
À N and TN removal rates
in M. aquaticum mesocosms with SW were approximately 169.5
and 157.8 mg N mÀ2
dÀ1
, respectively, which was comparable to the
Table 2
Nitrogen mass balance analysis in M. aquaticum mesocosms at the end of the experimental period.
Study wastewater
50% SW SW 200 NH4
þ
À N 400 NH4
þ
À N
Initial TN load (mass g-N) 3.44 6.87 3.00 6.00
Final TN load in wastewater
Mass g-N 0.14 0.32 0.42 1.34
Percentage of initial TN load (%) 4.22 4.65 13.9 22.3
Soil N accumulation
Mass g-N 0.95 1.13 1.11 2.63
Percentage of initial TN load (%) 28.0 16.6 37.0 43.8
Plant uptake
Mass g-N 1.06 1.08 1.38 1.44
Percentage of initial TN load (%) 31.2 15.9 46.2 24.1
Nitrification-denitrification
Mass g-N ~1.25 ~4.27 ~0.09 ~0.59
Percentage of initial TN load (%) 36.8 62.8 3.00 9.83
Fig. 4. The biomass (a) and total nitrogen content (b) of M. aquaticum harvested at the end of the experiment.
Table 3
Abundance of AOB, AOA, nirK, and nirS (gene copies gÀ1
dry soil) in M. aquaticum mesocosms with SW and 400NH4
þ
À N.
Time (day) Mesocosms with SW Mesocosms with 400 NH4
þ
À N
AOB AOA nirK nirS AOB AOA nirK nirS
0 2.96Eþ7d 2.37Eþ7b 4.59Eþ9c 1.66Eþ7d 3.13Eþ7a 2.43Eþ7b 4.61Eþ9a 1.58Eþ7b
7 4.56Eþ8a 3.44Eþ7a 4.12Eþ9c 2.57Eþ7c 2.03Eþ7b 1.58Eþ7c 2.32Eþ9c 7.61Eþ6c
14 3.77Eþ8b 3.28Eþ7a 6.45Eþ9b 5.42Eþ7b 2.34Eþ7b 1.51Eþ7c 3.67Eþ9b 4.65Eþ7a
28 1.08Eþ8c 2.73Eþ7ab 8.98Eþ9a 1.61Eþ8a 2.19Eþ7b 3.78Eþ7a 4.43Eþ9a 1.79Eþ7b
Data in the same column followed by the same letter have no significant differences at p 0.05.
F. Liu et al. / Journal of Environmental Management 166 (2016) 596e604 601

7. N removal rate of 162 mg N mÀ2
dÀ1
for a dairy wastewater
treatment wetland system vegetated with Typha latifolia and Typha
angustifolia (Gottschall et al., 2007).
4.2. Nitrogen conversion in M. aquaticum mesocosms
Low oxygen concentration is a limiting factor for NH4
þ
À N
oxidation in wetlands (Kouki et al., 2009), but plant ROL can pro-
vide the oxygen necessary to stimulate nitrification process and
promote N removal in a wetland matrix (Maltais-Landry et al.,
2009; Vymazal, 2013b). In the present study, the average DO
levels in M. aquaticum mesocosms were greater than 2 mg LÀ1
(Table 1), which provided sufficient oxygen for the transformation
of NH4
þ
À N to NO3
À
À N (Princic et al., 1998). Peak NO3
À
À N
concentrations were measured in M. aquaticum mesocosms with
swine wastewater on day 10; lower NO3
À
À N concentrations were
observed in synthetic wastewater during the study period (Fig. 1
(b)). Compared to M. aquaticum mesocosms with synthetic waste-
water, M. aquaticum mesocosms with swine wastewater had pH
values between 6.67 and 7.94 (Table 1), within the optimal range
for the nitrification process, i.e., 6.5e8.5 (Princic et al., 1998). These
results suggested that a strong nitrification process occurred in
M. aquaticum mesocosms with swine wastewater, resulting in sig-
nificant improvement in NH4
þ
À N and TN removal compared to
the mesocosms with synthetic wastewater (p 0.01). Schaafsma
et al. (2000) also found that the nitrification process in a con-
structed wetland markedly increased NH4
þ
À N removal from
ammonium-dominated dairy wastewater.
Denitrification may occur in the water-flooded soil layer con-
taining decaying litter material (Hadad et al., 2006), but the supply
capacity of bioavailable carbon limited the denitrification process in
wetlands (Maltais-Landry et al., 2009). Animal wastewater, which
characteristically contain high levels of organic carbon, provided
enough carbon to stimulate the denitrification process in wetlands
used for dairy wastewater treatment (Tanner et al., 1999), and
similar results were observed in wetlands used for swine waste-
water treatment (Hunt et al., 2002). After reaching a peak NO3
À
À N
concentration on day 10, the accumulated NO3
À
À N level in swine
wastewater in the present study decreased gradually until the end
of the incubation period. The reduction in NO3
À
À N concentration
indicated that denitrification was concurrent with nitrification in
M. aquaticum mesocosms containing swine wastewater. The
coupled nitrification and denitrification processes were necessary
for complete NH4
þ
À N removal in an ecological dairy wastewater
treatment system (Morgan and Martin, 2008). In the present study,
the coupled nitrification and denitrification in M. aquaticum mes-
ocosms contributed to the rapid NH4
þ
À N removal from swine
wastewater. In contrast, synthetic wastewater supplied no exoge-
nous carbon substrate, and the insufficient carbon was a limiting
factor for denitrification in M. aquaticum mesocosms with synthetic
wastewater.
4.3. Microbial processes in M. aquaticum mesocosms
The analysis of ammonia-oxidizing and denitrification microbes
showed that AOB was more abundant than AOA in the M. aquaticum
mesocosms with SW, suggesting that AOB may play a more
important role in nitrification process than AOA at higher nitrogen
loads (Di et al., 2009). Moreover, abundance of AOB in M. aquaticum
mesocosms with SW had a sharp increase on days 7, 14 and 28,
which was one order of magnitude higher than that on day 0. The
result also confirmed that AOB population distribution in CWs was
very sensitive to the variable environmental conditions (e.g. DO
level and ammonium concentration) (Gorra et al., 2007). In
M. aquaticum mesocosms with SW, the highest AOB abundance was
observed on day 7, and the decreased AOB abundance were
observed on days 14 and 28 (Table 3). The result indicated that the
changes of AOB abundance relating to ammonium oxidation pro-
cess were in combination with the NH4
þ
À N elimination pathway
(Fig. 1 (a)). The decreased AOB and AOA copy numbers were
detected in M. aquaticum mesocosms with 400 NH4
þ
À N on days 7,
14 and 28 compared to day 0, which indicated that synthetic
wastewater with high NH4
þ
À N concentrations inhibited the ac-
tivities of AOB and AOA responsible for ammonium oxidation
process. However, the markedly increased AOA copy numbers on
day 28 compared to day 0 implied that AOA were more sensitive in
response to the N variation than AOB (Ding et al., 2015), which
indicated AOA might play a more important role in the ammonium
oxidation process in M. aquaticum mesocosms with 400 NH4
þ
À N
after an incubation period of 14 days.
The abundance of nirK was two orders of magnitude higher than
nirS, suggesting that denitrifier harboring nirK may contribute to
more effects than nirS on the denitrification process in
M. aquaticum mesocosms. This result may be explained by that N-
rich ecosystems were beneficial to more nirK gene for denitrifica-
tion (Clark et al., 2012). Denitrifies were sensitive to the variation of
NO3
À
À N concentration, which was reflected by the markedly
increased abundance of nirK and nirS genes with the appearance of
high NO3
À
À N concentration on day 14. On day 28, the nirK gene in
M. aquaticum mesocosms kept increase while NO3
À
À N dis-
appeared, suggesting that the presence of plants in CWs may
stimulate the growth of denitrifies containing nirK gene (Chen
et al., 2014).
4.4. Nitrogen mass balance analysis
Plant nutrient uptake is influenced by the plant species, type of
wastewater, and nutrient loading rates (Gottschall et al., 2007). The
N balance analysis revealed that N uptake by M. aquaticum
accounted for 15.9e46.2% of the initial TN load in M. aquaticum
mesocosms with different wastewater treatments (Table 2), which
was comparable to the N accumulated by the plants (bulrushes,
bur-reed and cattail) by accounting for approximately 30% of the
wetland's annual nutrient budget (Hunt et al., 2002). For the
mesocosms with SW, N uptake by M. aquaticum accounted for 15.9%
of initial TN load, it indicated that plant uptake is a minor removal
path of N. Meanwhile, N uptake by M. aquaticum accounted for the
varied percentages of different N loads, which was consistent with
the reported results in a published paper (Gottschall et al., 2007).
Given that M. aquaticum can be reused as swine feed, harvesting
M. aquaticum would be a good practice for N removal from
wastewater and make the system more efficient for swine waste-
water treatment.
Less plant biomass with lower tissue N content was observed in
M. aquaticum mesocosms with swine wastewater compared to
synthetic wastewater (Fig. 4). This finding may be explained by the
following: (1) high-strength swine wastewater was assumed to
provide enough available N for M. aquaticum growth during the
early phase of the incubation period, but plant uptake may have
been overwhelmed by potent microbial nitrification and denitrifi-
cation in M. aquaticum mesocosms with swine wastewater; (2) very
low N concentrations in swine wastewater became a limiting factor
for plant growth and N uptake during the latter phase of the in-
cubation period.
Soil accumulation of TN accounted for 18.0e43.8% of the initial
TN load in M. aquaticum mesocosms. These findings were compa-
rable to those of Newman et al. (2000), who found that sediment
accumulation represented 10.6% and 35% of TN input to the wetland
in the first and second operation years, respectively. In
M. aquaticum mesocosms with swine wastewater, the N balance
F. Liu et al. / Journal of Environmental Management 166 (2016) 596e604602

8. analysis indicated that soil TN accumulation approximated that of
plant uptake, whereas soil TN accumulation accounted for a larger
proportion of initial TN load relative to plant uptake in
M. aquaticum mesocosms with high-strength synthetic wastewater
(400 NH4
þ
À N). Compared to synthetic wastewater, swine
wastewater with much dissolved organic carbon could increase the
amount of negative charge in soils, and therefore stimulated soil
NH4
þ
À N adsorption (Fernando et al., 2005). But soil accumulation
played a more important role in N removal from synthetic waste-
water than swine wastewater (Fig. 3 and Table 2), which may be
attributed to the faster NH4
þ
À N removal via nitrification and
denitrification in M. aquaticum mesocosms with swine wastewater.
The N mass balance analysis showed that the final TN load in
wastewater accounted for less than 5.05% of the initial TN load for
swine wastewater and approximately 22.3% for synthetic waste-
water. TN removal via nitrification and denitrification was esti-
mated to account for approximately 36.8% of the initial TN load for
50% SW and 62.8% for SW (Table 2). These findings indicate that
coupled nitrification and denitrification is an effective removal
pathway for N in M. aquaticum mesocosms with swine wastewater.
Similar results have been previously reported, indicating that
denitrification is a greater contributor to N removal in wetlands
than sediment adsorption or plant uptake (Maltais-Landry et al.,
2009). Nitrification and denitrification was negligible in
M. aquaticum mesocosms with synthetic wastewater, which may
have little effect on N removal from synthetic wastewater. Plant
uptake and soil accumulation were the major N removal mecha-
nisms in M. aquaticum mesocosms with synthetic wastewater.
A comprehensive understanding of the relativity and sustain-
ability of different N removal processes such as plant uptake,
sediment accumulation, volatilization, nitrification and denitrifi-
cation enable the improvement of N removal capabilities of con-
structed wetland systems (Tanner et al.,1995). Our study found that
plant N uptake and strong nitrification and denitrification occurred
in M. aquaticum mesocosms, which had a substantial impact on the
removal of NH4
þ
À N and TN from swine wastewater. M. aquaticum
may therefore be a good choice for the construction of wetlands to
treat animal wastewater. To really gain a deep understanding of N
cycle in these systems, the further experiments with multiple
loading batches and simultaneous microbial measurements are
needed in the future.
5. Conclusions
M. aquaticum was able to tolerate high-strength swine waste-
water with an NH4
þ
À N concentration of greater than 400 mg LÀ1
.
The removal rates of greater than 90% NH4
þ
À N and TN were
achieved in M. aquaticum mesocosms with swine wastewater. The
areal N removal rate was related to the N load; thus, M. aquaticum
mesocosms with high-strength swine wastewater achieved a
maximum areal TN removal of 157.8 mg N mÀ2
dÀ1
. Strong nitrifi-
cation and denitrification processes were speculated in
M. aquaticum mesocosms with swine wastewater, while they were
not deemed in M. aquaticum mesocosms with synthetic
wastewater.
The N mass balance analysis revealed that plant uptake and soil
accumulation were the main N removal pathways in M. aquaticum
mesocosms with synthetic wastewater, and nitrification and
denitrification contributed to a significant improvement in NH4
þ
À
N and TN removal in swine wastewater. These findings demon-
strate that M. aquaticum played an important role in N removal
from wastewater. However, given that this study focused on a single
batch addition to brand new wetlands, our outcome needs to be re-
confirmed by long-term experiments. Further works are therefore
needed to address the potential of M. aquaticum for the
improvement of long-term nutrient removal capability in full-scale
constructed wetlands.
Acknowledgments
This study was financially supported by the key CAS Program
(KZZD-EW-11-3), the CAS STS program (KFJ-EW-ZY-006), and the
National Science and Technology Supporting Project
(2014BAD14B01, 2014BAD14B05). We gratefully acknowledge
anonymous reviewers for their constructive comments and
suggestions.
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