Ecological Engineering 135 (2019) 45–53

Contents lists available at ScienceDirect

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

Effect of plant physiological characteristics on the removal of conventional T

and emerging pollutants from aquaculture wastewater by constructed
wetlands
Xiang-feng Huanga, Guang-yu Yea, Nai-kang Yia, Li-jun Lua, Lin Zhanga, Liu-yan Yangb, Lin Xiaob,
⁎
Jia Liua,
a
College of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Ministry of Education Key Laboratory of Yangtze River
Water Environment, Shanghai Institute of Pollution Control and Ecological Security, Tongji University, Shanghai 200092, China
b
State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental, Nanjing University, Nanjing 210023, China

A R T I C LE I N FO A B S T R A C T

Keywords: Four horizontal subsurface flow pilot-scale constructed wetlands (CWs) named as S1, S2, M1 and M2 were
Constructed wetlands constructed to treat aquaculture wastewater. And two different plant species (Iris pseudacorus and Phragmites
Plant root system australis) were cultivated in single and mixed planting patterns in these four CWs. The removal rate of con-
Antibiotics ventional pollutants (nutrient and organic compounds), antibiotics including enrofloxacin (ENR), sulfa-
Antibiotic resistance genes
methoxazole (SMZ), and antibiotic resistance genes (ARGs) were evaluated among those CWs. The total nitrogen
Microbial community
and NH4+–N removal rates of all CWs were 73.24%–91.46% and 61.20%–92.27%, respectively. CWs with mixed
planting patterns, such as M1 (planted with Iris pseudacorus at the forepart and Phragmites australis at the back)
and M2 (alternate cultivation with Iris pseudacorus and Phragmites australis) showed better performances than
CWs planted with single plant species, such as S1 (Iris pseudacorus) and S2 (Phragmites australis). However, S1
and S2 exhibited higher removal efficiencies for emerging contaminants: S1 had removal efficiencies of 77.64%,
68.70%, and 58.21% for ENR, SMZ, and total ARGs, respectively, and S2 had removal efficiencies of 81.11%,
64.94%, and 56.26% for ENR, SMZ, and total ARGs, respectively. Compared with single planting, the dominant
genera in mixed planting exhibited lower relative abundance in anaerobes and higher percent of bacteria as-
sociated with nitrogen metabolism, indicating that different plant physiological characteristics affected the
microbial community structures of the CWs.

1. Introduction
Aquaculture has been a growing industry due to its socio-economic
benefits, sustainable development and potential to alleviate the world
hunger (Ling et al., 2004). However, aquaculture wastewater can cause
severe environmental problems because of its high discharge amounts,
high chemical oxygen demand (COD), and high nitrogen and phos-
phorus contents (Ling et al., 2004). Meanwhile, antibiotics are widely
used in aquaculture to prevent infectious diseases among aquaculture
species. Although antibiotics exist at low concentrations in aquatic
environments, their persistence can promote the development of bac-
teria with antibiotic-resistant genes (ARGs) as a result of selective
pressure (Fernandes et al., 2015; Michael et al., 2013; Cabello, 2006).
This consequence can reduce the therapeutic potential of antibiotics
against human (Heuer et al., 2009) and animal pathogens (Michael
et al., 2013), and change bacterial flora in sediments and water columns
(Cabello, 2006). Therefore, the removal of conventional and emerging
pollutants from high-density aquaculture wastewater should receive
more attention.
As a treatment technology for aquaculture wastewater, constructed
wetlands (CWs) have the advantage of low investment requirements,
implementation costs, and energy consumption (Meng et al., 2014;
García et al., 2010). Previous studies demonstrated that CWs were
capable of removing a majority of environmental pollutants including
nitrogen, phosphorous, COD (Wang et al., 2013; Li et al., 2013; Hu
et al., 2012; Lin et al., 2002), emerging contaminants, such as anti-
biotics (Berglund et al., 2014; Chen et al., 2015; Hijosa-Valsero et al.,
2011; Liu et al., 2013) and ARGs (Chen et al., 2015; Liu et al., 2013).
These advantages are attributed to the interactions among soil/sedi-
ment, plants and microorganisms (Carvalho et al., 2012), where

⁎
Corresponding author.
E-mail address: liujia@tongji.edu.cn (J. Liu).

https://doi.org/10.1016/j.ecoleng.2019.05.017
Received 22 December 2017; Received in revised form 13 May 2019; Accepted 17 May 2019
Available online 23 May 2019
0925-8574/ © 2019 Elsevier B.V. All rights reserved.
X.-f. Huang, et al.
multiple physical, chemical and biological processes occur simulta-
neously, like adsorption, photolysis, volatilization, plant uptake and
accumulation, plant exudation and microbial degradation (Garcia-
Rodríguez et al., 2014; Vymazal et al., 2010). Therefore, the perfor-
mance of pollutant removal of CWs is dependent on design parameters,
which include plant species, flow type, substrate, hydraulic loading
rates, hydraulic retention time (HRT), and applied pollutant loadings
(Hijosa-Valsero et al., 2011; Wu et al., 2015, 2014; Weerakoon et al.,
2013; Hijosa-Valsero et al., 2010).
The presence of macrophytes distinguishes CWs from unplanted soil
filters or lagoons (Vymazal, 2011). Many studies have identified plants
and microorganisms associated with plant roots contributed the CWs
performance (Bouali et al., 2014). The belowground parts of plants in
CWs can provide adsorption sites for bacterial, release oxygen and root
exudates to the rhizosphere, which is beneficial for the growth of mi-
croorganism (Hijosa-Valsero et al., 2011; Vymazal, 2011a, 2011b).
Therefore, CWs cultivated with different plant species have different
influence on the removal efficiencies of pollutants. In the past few
years, studies have mainly focused on the effects of single plant species
on the treatment performance of CWs, but in recent years, more at-
tention has been paid to the effects of mixed planting using plants with
different physiological characteristics. In our previous study, we found
that CWs planted with Iris pseudacorus had higher purification effi-
ciency for nitrogen in aquaculture wastewater than Phragmites australis
(Huang et al., 2016). Other studies have reported the advantages of
Phragmites australis in the removal of antibiotics and ARGs (Carvalho
et al., 2012; Liu et al., 2013; Choi et al., 2016). Given the emerging
pollutant characteristics of aquaculture wastewater, this study aimed to
explore the effects of different plant physiological characteristics (plant
species and planting patterns) of four pilot-scale CWs on the removal
efficiencies for conventional and emerging pollutants, such as anti-
biotics and ARGs. In addition, the microbial community structures of
the four pilot-scale CWs were characterized through quantitative
polymerase chain reaction (qPCR) assays and high-throughput se-
quencing.
2. Materials and methods
2.1. Experimental design and operation methods
These wetland microcosms were located at an aquaculture farm of
yellow catfish in Changzhou (31°35′N, 119°52′E), Jiangsu Province,
China. The size of four horizontal subsurface-flow constructed wetlands
(HSSFCW) was 150 cm long, 40 cm wide, and 80 cm deep (effective
depth of 60 cm), and the materials were polyvinyl chloride. The two
ends of the microcosm were separated by perforated plates, forming a
15 cm water distribution area and a 15 cm water collection area. Both
areas were filled with 40 cm-depth gravel (particle diameter of
30–50 mm). Previous study had investigated the filler species and filling
pattern, and found that zeolite and gravel performed better in the
pollutants removal. The filling pattern of treatment area was gravel
(particle diameter of 5–10 mm) filling in the front three-quarters area
near the inlet and zeolite (particle diameter of 5–15 mm) filling in the
back quarter area near the outlet (Huang et al., 2016). Therefore, these
two plants commonly planted in CWs were selected for this experiment.
As shown in Fig. 1, different plant species in different planting patterns
were cultivated in each CW. S1 was planted with Iris pseudacorus, and
S2 was planted with Phragmites australis. In M1, Iris pseudacorus and
Phragmites australis were separately planted at the forepart and back at
a 1:1 ratio. In M2, Iris pseudacorus and Phragmites australis were ar-
ranged alternately. Planting density of each wetland was 50 plants
m−2. The influent that fed the CWs was peristaltically pumped from
one of the fish ponds of the aquaculture farm. HRT and hydraulic load
were three days and 0.084 m3 m−2 d−1, respectively. The whole system
was initiated in September 2014 and ran continuously. The data ana-
lysis in this work started in June 2016 and ended in September 2016.
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Ecological Engineering 135 (2019) 45–53
2.2. Samples collection
In the wastewater sampling campaign, we collected and monitored
the influent and effluent samples of the four CWs once per 4 days. The
wastewater samples for analysis of conventional contaminant, anti-
biotics and ARGs were collected from the bucket and water outlets in 1-
L polyethylene plastic bottle.
In the substrate sampling campaign, we selected 4 points (the upper
front (UF), the upper back (UB), the lower front (LF) and the lower back
(LB)) as the sampling sites. The front sampling point was set at 30 cm
from the water distribution area, the back sampling point was set at
30 cm from water collection area, and vertical sampling points were at
the depth of 10 cm and 50 cm, respectively. After the two years of stable
operation of the CWs, a portion (30 g) of the substrate from each
sampling point was obtained using sterile spatula once in July and once
in August 2016. All the samples were immediately frozen at −20 °C
prior to laboratory analysis.
2.3. Chemical analysis
2.3.1. Physicochemical parameters and conventional wastewater quality
parameters
Collected water samples were analyzed for temperature, dissolved
oxygen (DO), pH, chemical oxygen demand (COD), total phosphorus
(TP), total nitrogen (TN), NH4+–N, NO3−–N, and NO2−–N in ac-
cordance with the Standard Methods for Monitoring Water and
Wastewater Quality (State Environmental Protection Administration,
2002). To maintain the samples in an equilibrium state and to obtain a
more reliable estimation of the removal efficiency, the water quality
parameters were measured immediately after sampling.
2.3.2. Extraction and determination of antibiotics and quantification of
ARGs
The antibiotic analysis of the water samples was conducted based on
our previous studies (Liu et al., 2016). Enrofloxacin (ENR) and sulfa-
methoxazole (SMZ) are two common antibiotics used in the aquaculture
farms. Liquid chromatography in combination with tandem mass
spectrometry (LC–MS/MS) was used to analyze the concentrations of
ENR and SMZ in water samples.
The quantification ARGs of influent, effluent and substrates of the
four CWs were commissioned to Shanghai Kai Biotechnology Co, Ltd
and their average ARG content was obtained. A total of 40 cycles was
performed to improve the chances of product formation from low initial
template concentrations. A 10 µL PCR reaction solution was employed
and contained 5 µL Roche FastStart Universal SYBR Green Master
(ROX), 3 µL ddH2O, 0.75 µL forward primer (30 µM), 0.75 µL of each
primer (30 µM), and 0.5 µL DNA sample. qPCR assays were run on an
Applied Biosystems 7500 Fast Real-Time PCR System (ABI, USA). The
temperature program for the quantification of ARGs consisted of initial
denaturation at 95 °C for 10 min, followed by 40 cycles for 30 s at 95 °C
and at 60 °C for 30 s.
2.4. Quantitative and high-throughput sequencing analysis of microbial
communities
2.4.1. DNA extraction
After two year of stable operation, SoilDNA Kit (E.Z.N.A.®, Omega
Bio-tek, Norcross, GA, U.S.A.) was used to extract total genomic DNA
from the substrate samples described in Section 2.2. The quantity and
quality of the extracted DNA were examined by measuring their ab-
sorbance at 260 and 280 nm using UNICO-2100 UV/VIS spectro-
photometer.
2.4.2. Real-time PCR assays
Real-time PCR assays were conducted to quantify the total bacterial
16S rRNA using primers 338f and 518r. qPCR was performed using a
X.-f. Huang, et al. Ecological Engineering 135 (2019) 45–53

Fig. 1. Schematic of the pilot-scale constructed wetlands. Blue arrows indicate the wastewater flow path, and the brown layers indicate gravel and zeolite shell.
represents the substrate sampling sites for ARGs and microbial communities. The UF means substrate of upper front, UB means substrate of upper back, LF means
substrate of lower front, LB means substrate of upper back.

two-step amplification procedure. The thermal cycling conditions
comprised 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and
60 s at 60 °C. All samples were run in triplicate. A standard curve was
calculated using duplicate ten-fold dilution series of standard DNA.
2.4.3. High-throughput sequencing and analysis
The bacteria 16S ribosomal RNA genes were amplified by PCR (at
95 °C for 3 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s,
72 °C for 45 s, and a final extension at 72 °C for 10 min) using primers
338f and 803r, where the barcode is an eight-base sequence unique to
each sample. The. PCR reactions were performed in triplicate in a 20 µL
mixture containing 4 µL of 5 × FastPfu Buffer, 2 µL of 2.5 mM dNTPs,
0.8 µL of each primer (5 M), 0.4 µL of FastPfu Polymerase, and 10 ng of
template DNA. Amplicons were extracted from 2% agarose gels and
purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences,
Union City, CA, U.S.) following the manufacturer’s instructions, and
quantified using QuantiFluorTMST (Promega, U.S.A.). Purified ampli-
cons were pooled in equimolar and were pair-end sequenced (2 × 250)
on an Illumina MiSeq platform according to the standard protocols.
Raw FASTQ files were demultiplexed and quality-filtered using QIIME
(version 1.17) as follows: (i) The 300-bp reads were truncated at any
site that received an average quality score < 20 over a 50-bp sliding
window, and the reads that were shorter than 50 bp were discarded. (ii)
The barcodes had to match exactly, reads with two nucleotide mis-
matches in the primer matching or containing ambiguous characters
were removed. (iii) Only sequences that overlapped for more than 10 bp
were assembled according to their overlap sequence. Reads that could
not be assembled were discarded. Operational units (OTUs) were
clustered with a 97% similarity cut-off using UPARSE (version 7.1
http://drive5.com/uparse/). Chimeric sequences were identified and
removed using UCHIME.
2.5. Calculation of removal efficiencies and statistical analysis
The removal efficiency (%) of contaminants and antibiotics were
calculated using the formula (1 − Ce/Ci) × 100%, where Ci and Ce
(mg/L) indicate the concentration of target compounds in the influent
and effluent respectively. In order to clearly quantify the content of all
ARGs, the content of various ARGs were summed and presented with
Σqep, Σsul, Σtet and ΣARGs. The removal efficiency (%) of ARGs were
calculated using the formula (1 − ΣACe/ΣACi) × 100%, where ACi and
ACe (mg/L) indicate the absolute concentration of each ARG in the
influent and effluent respectively, ΣACe and ΣACI indicate the sum of all
genes of a particular ARG-family in the influent and effluent respec-
tively.
Data calculation and transformation were carried out using
Microsoft Excel 2016. One-way ANOVA with Duncan test was used to
evaluate the statistical significance of difference using SPSS version 22.
3. Results
3.1. Operational performance of the four CWs
The conventional wastewater parameters (DO, COD, TN, NH4+–N,
NO3––N, NO2––N, and TP) of the influent and effluent samples were
summarized in Table 1. In the influent, DO, COD, TN, NH4+–N,
NO3––N, NO2––N and TP were detected at the average concentrations of
2.45, 66.6, 3.60, 2.35, 0.51, 0.134 and 0.23 mg/L, respectively. In the
effluents (S1, S2, M1, M2), these parameters were declined to
0.97–1.14, 30.7–36.5, 0.59–0.77, 0.39–0.52, 0.05–0.08, 0.001–0.003

Table 1
Average of conventional quality parameters of aquaculture wastewater in the four CWs (n = 3).
Conventional quality parameters (mg/L)

DO COD TN 4 –N
NH+ NO−
3 –N NO−
2 –N TP

Influent 2.45 ± 0.13 66.6 ± 6.44 3.60 ± 1.31 2.35 ± 0.56 0.51 ± 0.013 0.134 ± 0.03 0.23 ± 0.047
S1 0.97 ± 0.08 36.5 ± 3.36 0.71 ± 0.13 0.53 ± 0.19 0.06 ± 0.0021 0.003 ± 0.00 0.06 ± 0.0042
S2 1.14 ± 0.11 30.7 ± 2.19 0.77 ± 0.16 0.45 ± 0.08 0.08 ± 0.0045 0.002 ± 0.00 0.04 ± 0.0025
M1 1.05 ± 0.08 35.4 ± 3.60 0.59 ± 0.19 0.39 ± 0.11 0.05 ± 0.0083 0.001 ± 0.00 0.07 ± 0.0120
M2 1.02 ± 0.12 31.7 ± 4.47 0.59 ± 0.18 0.39 ± 0.12 0.06 ± 0.014 0.001 ± 0.00 0.06 ± 0.0086

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Fig. 2. The removal rate of TN (a) and NH4+–N (b) by the four CWs with single vegetation (S1 planted with Iris pseudacorus and S2 planted with Phragmites australis)
and mixed vegetation (M1 planted with Iris pseudacorus at the forepart and Phragmites australis at the back at a 1:1 ratio, M2 planted with Iris pseudacorus and
Phragmites australis alternately). Note that scales on the Y-axes are different between the two figures.

and 0.04–0.07 mg/L, respectively. The average concentrations of DO
and conventional contaminants significantly decreased, and the re-
moval rates of contaminants ranged from 43.6% to 99.16%, indicating
that the four CWs had distinct effects on the removal of conventional
pollutants. Regarding to the nitrogen pollutant of the aquaculture
wastewater, the removal rates of M1 and M2 for TN and NH4+–N were
higher than those of S1 and S2 (Fig. 2). In contrast, S2 reached the
highest removal rate of COD at 53.9%, followed by M2, M1 and S1.
and SMZ were 77.6% and 68.7% for S1, 81.1% and 64.9% for S2, 75.7%
and 59.2% for M1, and 75.6% and 54.3% for M2. The removal rates of
the four CWs for ENR was higher than that for SMZ. Moreover, plant
species and planting patterns showed significant effects on antibiotic
removals (p < 0.05). The removal rates for ENR and SMZ were in the
following order: S2 > S1 > M1 > M2 and S1 > S2 > M1 > M2.
3.3. Abundance and removal of ARGs in the four CWs

3.2. Removal of antibiotics by the four CWs
The concentrations of antibiotics in the influent and effluent of the
four CWs are shown in Fig. 3. The influent ENR and SMZ concentrations
were 25.7–67.4 ng/L and 64.0–211.2 ng/L, respectively. These broad
ranges were associated with the temporal variation of antibiotic dosage
in the fish pond. The average ENR concentrations in effluents from S1,
S2, M1, and M2 were 9.96, 8.25, 10.51, and 10.48 ng/L, respectively.
The average SMZ concentrations in effluents from S1, S2, M1, and M2
were 41.47, 46.32, 51.13, and 61.14 ng/L for SMZ, respectively. In
terms of removal efficiency (Fig. 4), the average removal rates of ENR
Eleven ARGs with higher content in influent, effluent and substrates
were showed in Figs. 5a and 6, including four quinolone resistance
genes (qepA, qnrA, qnrB, and qnrD), two sulfonamide resistance genes
(sul1 and sul3), and five tetracycline resistance genes (tet(34), tetB,
tetL, tetM, and tetV). Among the three classes of target ARGs in was-
tewaters and substrates, quinolone resistance genes were the most
abundant, followed by sulfonamide resistance genes and tetracycline
resistance genes. With regard to individual genes, qepA, qnrA, qnrD,
sul1 and tet(34) were dominant in the influent, while the most abun-
dant genes in the substrates were qepA, qnrA and sul1.
Compared to the influent, the absolute content of ARGs in effluent

Fig. 3. Concentrations of detected enrofloxacin (a) and sulfamethoxazole (b) in the influent and effluent of the four CWs (n = 3) with single vegetation (S1 planted
with Iris pseudacorus and S2 planted with Phragmites australis) and mixed vegetation (M1 planted with Iris pseudacorus at the forepart and Phragmites australis at the
back at a 1:1 ratio, M2 planted with Iris pseudacorus and Phragmites australis alternately). The error bars represent standard deviation (n = 3).

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X.-f. Huang, et al. Ecological Engineering 135 (2019) 45–53

Fig. 4. The average removal rate of enrofloxacin (a) and sulfamethoxazole (b) in the influent and effluent by the four CWs with single vegetation (S1 planted with Iris
pseudacorus and S2 planted with Phragmites australis) and mixed vegetation (M1 planted with Iris pseudacorus at the forepart and Phragmites australis at the back at a
1:1 ratio, M2 planted with Iris pseudacorus and Phragmites australis alternately). The error bars represent standard deviation (n = 3).

decreased significantly. The average removal rates for ∑qepA, ∑sul, and
∑tet were 57.0%, 54.9%, and 80.9% by S1, 54.8%, 65.5%, and 67.0%
by S2, 34.9%, 42.1%, and 53.2% by M1, and 53.5%, 62.9%, and 83.7%
by M2. In the four CWs, the removal rate of ∑tet was higher than those
of ∑qepA and ∑sul. In general, the aqueous removal rates of all target
ARGs (∑ARGs) ranged from 36.5% to 58.2%. The removal efficiencies
for ∑ARGs by the four CWs had the following order:
S1 > S2 > M2 > M1 (Fig. 5b).
As shown in Fig. 6, the absolute content (average of the content of
the four substrate sites) of qepA, qnrA, and sul1 in substrate were
4.62 × 108, 6.68 × 106, and 5.85 × 106 copies/g for S1, 3.21 × 108,
2.43 × 107, and 1.17 × 107 copies/g for S2, 2.30 × 108, 1.92 × 107,
and 1.14 × 107 copies/g for M1, and 4.67 × 108, 3.86 × 107, and
3.54 × 107 copies/g for M2. The absolute content of ARGs was lowest
in the substrate of M1, while showed the highest in the substrate of M2.
Different kinds of ARGs performed different residual content in the four
CWs.
3.4. Structure of the microbial community in the four CWs
The OTU numbers, Shannon index, Chao index, ACE index, and
Simpson index were calculated using the diversity formula. OTU (op-
erational taxonomic units) is artificially set for a taxonomic unit in
order to facilitate analysis in phylogenetic or population genetics stu-
dies. Through the classification operation, the sequences are classified
into various groups according to their similarity. The number of OTUs
can represent the diversity of the population to some extent. Chao Index
and the Ace Index represent the estimate number of OTUs contained in
a sample, which can be used to characterize the abundance of microbial
populations. Both the Shannon index and the Simpson index estimate
the microbial diversity index in the sample. The larger Shannon index
and lower Simpson index often indicate the higher community di-
versity. As shown in Table 2, the average OTU numbers, Ace index and
the Chao index were 2199, 2729 and 2757 for S1, 2311. 2808 and 2841
for S2, 2654, 3225 and 3225 for M1, and 2389, 2985 and 2985 for M2.
The OUT, Ace index and Chao index in the substrates of the four CWs
were in the following order: M1 > M2 > S2 > S1. Shannon index
showed some differences with other index. But the statistical analysis
showed that the microbial diversity and abundance were significantly
higher under mixed planting than that under single planting
(p < 0.05).
With the help of 454-pyrosequencing, the relative abundances of

Fig. 5. Absolute content (a) of ARGs in the influent and effluent of the four CWs and removal rate (b) of ARGs by the four CWs with single vegetation (S1 planted with
Iris pseudacorus and S2 planted with Phragmites australis) and mixed vegetation (M1 planted with Iris pseudacorus at the forepart and Phragmites australis at the back at
a 1:1 ratio, M2 planted with Iris pseudacorus and Phragmites australis alternately).

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Denitratisoma, which were different with that in S1 and S2. It was

worthy to be noted that the relative abundance of anaerobes was lower
and the percent of bacteria associated with nitrogen metabolism was
higher in mixed planting, which might be the reason for the higher
removal efficiency of nitrogen pollutants under mixed planting condi-
tions. But the detailed mechanism needs a further research.

4. Discussion

4.1. Operational performance

Regarding to the removal rates of COD, the difference of removal

Fig. 6. Absolute content of ARGs (average content of the four substrate sam-
pling sites) in the substrate of the four CWs with single vegetation (S1 planted
with Iris pseudacorus and S2 planted with Phragmites australis) and mixed ve-
getation (M1 planted with Iris pseudacorus at the forepart and Phragmites aus-
tralis at the back at a 1:1 ratio, M2 planted with Iris pseudacorus and Phragmites
australis alternately).
bacterial taxa were examined at different levels to determine whether
there were significant differences in the compositions of the bacterial
communities in the four CWs. The bacterial community compositions of
samples from the four microcosms on the phylum and genus levels were
presented in Figs. 7 and 8. The figures illustrated that Proteobacteria,
Bacteroidetes, Chloroflexi, Acidobacteria, and Actinobacteria were the
most dominant phyla in all samples, but their distributions were rela-
tively different. On genus level, Anaerolineaceae, Lysobacter, Thiobacillus
and Acidobacteria were the dominant genera in S1 and S2. The percent
of Anaerolineaceae in S1 was higher than that in S2, and the percent of
aerobes such as Lysobacter and Nitrospira was lower in S1, which might
be attributed to the more root oxygen release of Phragmites australis and
contributed to the higher removal efficiency of COD and NH4+–N in S2
(Wang et al., 2012). However, the dominant genera in M1 and M2 were
Anaerolineaceae, Nitrospira, Nitrosomonasdace, Hydrogenophilace and
rate between S1, S2, M1 and M2 wasn’t significant (P > 0.05). S2
planted with Phragmites australis achieved the highest removal rate,
which is consistent with the result of Wang (Wang et al., 2012). The
belowground biomass of Phragmites australis is well-developed, which is
positively correlated to radial oxygen loss (ROL) and stimulates sub-
strate respiration rate and phosphatase activity (Wang et al., 2009;
Wießner et al., 2002; Li et al., 2009). As a result, the COD removal was
enhanced in S2. Wu’s study also found that the removal rate of COD was
consistent with the ROL of plant roots (Wu et al., 2010). Lin’s study
compared ROL of eight wetland plants and found that Iris pseudacorus
had the minimum ROL, which was only 68.81 μmol O2/(d·g (DWroot))
(Lin et al., 2015). Besides, S2 also performed highest removal rate of
TP, which was consistent with the result of COD. The underground
rhizomes of the Phragmites australis are criss-crossed and form a large
underground root system, which is beneficial to the interception and
removal of phosphorus (Li et al., 2009). The study of Yin also proved
that the wetland planted with Phragmites australis had a high removal
effect on phosphorus, which was consistent with this study (Wang and
Yin, 2000).
The removal rates of TN and NH4+–N by M1 and M2 were higher
than those by S1 and S2. These results suggest that mixed vegetation is
more efficient in nitrogen pollutant removal than single species, which
is in agreement with those of other studies (Fraser et al., 2004). In a
complicated system of CWs with mixing plants pattern, several nitrogen
removal pathways contribute to the purification efficiency. On one
hand, 5%–30% of nitrogen can be removed by plant uptake (Maltais-
Landry et al., 2009; Li et al., 2009), Our previous study found that the
nitrogen content in per unit mass of Iris pseudacorus and Phragmites
australis was 25.9 mg/g and 15.3 mg/g, respectively (Huang et al.,

Table 2
Diversity index and evenness of the different positions in the four CWs. Values are given as mean ± standard deviation (n = 3).
Sample ID OTU Ace index Chao index Shannon index Coverage rate

S1 UF 1960 ± 19.92 2480 ± 15.05 2564 ± 38.88 6.13 ± 0.0031 0.97949

UB 1894 ± 24.83 2349 ± 13.71 2375 ± 29.53 5.89 ± 0.018 0.98291
LF 2410 ± 17.75 2996 ± 19.31 2959 ± 23.17 6.72 ± 0.015 0.97189
LB 2532 ± 37.37 3091 ± 20.69 3129 ± 47.89 6.60 ± 0.042 0.97374
Average 2199 ± 23.97 2729 ± 14.94 2757 ± 30.24 6.34 ± 0.025 /

S2 UF 2160 ± 31.80 2681 ± 14.17 2675 ± 21.40 6.28 ± 0.051 0.98262

UB 1957 ± 21.59 2473 ± 11.87 2479 ± 27.96 5.89 ± 0.029 0.97911
LF 2604 ± 19.09 3072 ± 14.83 3145 ± 18.48 6.83 ± 0.012 0.98046
LB 2523 ± 17.31 3004 ± 10.01 3065 ± 35.55 6.75 ± 0.009 0.97769
Average 2311 ± 13.43 2808 ± 9.58 2841 ± 20.38 6.44 ± 0.014 /

M1 UF 2376 ± 15.87 2874 ± 15.55 2937 ± 28.67 6.56 ± 0.024 0.97876

UB 2925 ± 23.65 3539 ± 19.10 3533 ± 17.58 6.94 ± 0.072 0.97559
LF 2734 ± 32.55 3218 ± 16.26 3229 ± 35.13 6.77 ± 0.11 0.98293
LB 2581 ± 22.58 3190 ± 14.78 3201 ± 19.33 6.39 ± 0.023 0.9747
Average 2654 ± 19.46 3205 ± 10.96 3225 ± 24.18 6.67 ± 0.018 /

M2 UF 2014 ± 26.93 2482 ± 16.56 2454 ± 29.02 5.33 ± 0.079 0.98305

UB 2402 ± 18.46 3032 ± 19.34 3046 ± 13.29 5.88 ± 0.041 0.98039
LF 2762 ± 15.40 3281 ± 22.86 3287 ± 20.01 6.91 ± 0.019 0.97667
LB 2377 ± 23.44 3160 ± 25.43 3154 ± 30.84 6.30 ± 0.022 0.96937
Average 2389 ± 17.12 2989 ± 15.61 2985 ± 28.99 6.11 ± 0.036 /

UF = upper front substrates, UB = upper back substrates, LF = lower front substrates, LB = lower back substrates.

50
X.-f. Huang, et al.
Fig. 7. Phylum level compositions and distribution of bacteria community in the CWs with single vegetation (a) and mixed vegetation (b).
Fig. 8. Genus level compositions and distribution of bacteria community in the CWs with single vegetation (a) and mixed vegetation (b).
2016). Wang’s study also found that the accumulation of nitrogen in Iris
(326.3 g/m2) was higher than that in Phragmites australis (131.9 g/m2)
(Wang et al., 2016). On the other hand, the intensity of nitrification and
denitrification plays a critical role in nitrogen removal, which is con-
siderably affected by root oxygen release and exudates (Maltais-Landry
et al., 2009). The two plants releases different amounts of oxygen to the
environment (Wang et al., 2012), which form anaerobic and aerobic
alternating environment. In conclusion, the higher nitrogen removal
rate of mixed planting may be attributed to the cooperative function of
the two plants.
4.2. Removal of antibiotics
Removal of antibiotics in aquaculture wastewater can be achieved
through various degradation mechanisms, including physicochemical
degradation, photodegradation (photolysis), biodegradation (microbial
activity), and adsorption to the wetland substrate. The removal me-
chanism generally depends on the physicochemical properties of anti-
biotics (Choi et al., 2016). As a kind of fluoroquinolones, ENR has two
six-membered rings of quinolones combined with fluorine atoms, which
results in the low biodegradation rate, high photolysis and adsorption
rate of ENR (Paul et al., 2007; Conkle et al., 2010). Although photolysis
is negligible in HSSFCW, zeolite and gravel particles with large surface
areas, as well as the abundant calcium, magnesium, and other metal
51
Ecological Engineering 135 (2019) 45–53
ions on the surfaces of these particles, can form ligand complexes with
the carboxyl and carboxide groups of ENR. Therefore, it can be con-
cluded that sorption plays a significant role to remove ENR in HSSFCW
(Huang et al., 2016). Compared with Iris pseudacorus; Phragmites aus-
tralis with deeper and denser root system enlarges the area for biofilm
growth (Wang et al., 2012), clogs substrate pores, and enhances ENR
adsorption. These effects collectively contribute to the higher ENR re-
moval rate of S2 than that of S1.
On the contrary, SMZ, a kind of sulfonamides, has high mobility and
low sorption to a solid substrate (Liu et al., 2013; Chen et al., 2011).
Previous study implies that there are no sulfonamides detected in the
substrate or plant sample, and degradation (and transformation) plays a
more significant role in the aqueous removal of sulfonamides than
substrate adsorption and plant uptake (Chen et al., 2016). In addition,
anaerobic conditions and low oxidation–reduction potential (ORP) ac-
celerate the fermentation and degradation of SMZ (Carballa et al.,
2006). As shown in Fig. 8, the relative abundances of Anaerolineaceae
were generally in the order of S1 > S2 > M1 > M2 and corre-
sponded with that of the removal rate of SMZ. This result suggests that
the degradation of microbial community in CWs has effects on the re-
moval of SMZ, which is in accordance with the result of Jia’s study (Jia
et al., 2012). Therefore, it can be reasonably concluded that anaerobic
pathway is an important degradation pathway for SMZ. In addition,
organic matter and root exudate are found to accelerate the degradation
X.-f. Huang, et al.
and removal of SMZ (Li et al., 2017). As described above, Iris pseuda-
corus, which has slower oxygen loss and more root exudates than
Phragmites, not only reduces the ORP but also increases the content of
small-molecule organic matter in the CWs. Exudates have crucial effects
on the root microenvironment, thus providing a better environment for
the microbial community to degrade or transform SMZ. Compared with
single planting, the interaction between the root exudates of Iris pseu-
dacorus and the oxygen release of Phragmites australis in mixed planting
enriches microbial diversity (Table.2), which induces competition
among microbes. Consequentely, the abundance and activity of the
microbial community that can degrade SMZ decrease, which may re-
duce the removal efficiency of SMZ.
4.3. Removal of ARGs
As biological contaminants, ARGs exhibit different behaviors and
fates in various environmental media because of biological activities
(Auerbach et al., 2007). ARGs can be transferred among microbes
through typical gene capture and dissemination systems called in-
tegrons, which are prevalent in environments affected by anthro-
pogenic activities (Chen et al., 2015). The biological process in CWs has
a complex role in ARGs removal because it may lead to ARGs trans-
mission and proliferation or may be involved in ARGs degradation
(Ghosh and Lapara, 2007; Guo et al., 2014; Ying et al., 2014). The large
amounts of attachment sites, rapid oxygen loss, and exudates of dense
roots in CWs provide an optimal environment for the expansion of
microbial communities and populations in sediments and wastewater,
subsequently promoting the dissemination of ARGs via horizontal gene
transfer among different bacterial species and vertical gene transfer
within the same microbial species that carry ARGs (Fang et al., 2017;
Zhang et al., 2017). In this study, the order of the absolute ARGs con-
centrations in effluent was M1 > M2 > S2 > S1. This order corre-
sponded with the microbial diversity of the four CWs. The microbial
diversity and absolute abundance of S1, which was only planted with
Iris pseudacorus, were lowest due to its poorer root microenvironment
than S2 planted with Phragmites australis. Thus, the effluent from S1 had
the lowest ARGs concentration. In contrast, the combination of the
vigorous root exudation of Iris pseudacorus and the affluent oxygen re-
lease of Phragmites australis provide a more appropriate growth en-
vironment for different bacteria in both the front and the end of M1.
This environment contributes to the highest diversity of the microbial
community and absolute ARGs content in the effluent from M1.
In HSSFCW, the host bacteria that carry ARGs are intercepted by the
substrate and are transferred from the aqueous phase to the solid sur-
face. If not completely degraded, ARGs will be re-discharged to the
environment with the effluent, transforming the CWs into potential
pollution sources of ARGs (Huang et al., 2017). Some study indicates
that ARGs quantities exhibit statistically significant reductions in an
anaerobic environment and ORP is a critical role in the biodegradation
of ARGs in CWs and is affected by the radial oxygen loss of different
plants (Dušek et al., 2008); but the removal process and mechanism of
CWs for ARGs requires further investigation
5. Conclusion
The different removal performances of CWs planted with two plant
species and two planting patterns might be attributed to the plant
physiological characteristics and their rhizosphere microenvironments.
Mixed planting improved the removal of TN and NH4+–N in the
aquaculture wastewater through the cooperation between the nutrient
uptake of Iris pseudacorus and the enhanced nitrification efficiency of
Phragmites australis. The vigorous root exudate of Iris pseudacorus and
the oxygen release of Phragmites australis increased the diversity and
competition of microbial communities in CWs, which subsequently
decreased the removal rate of SMZ and ARGs. Consequently, plant
species and planting patterns should be properly designed considering
52
Ecological Engineering 135 (2019) 45–53
the wastewater characterization and plant physiological characteristics,
which would benefit the joint removal of complex pollutants.
Verification in a larger pilot scale as well as the efficient management
might be favorable for the future study.
Acknowledgments
This work was supported by the Major Science and Technology
Program for Water Pollution Control and Treatment
(2017ZX07204002, 2017ZX07204004, 2012ZX07101006) and
Fundamental Research Funds for the Central Universities
(0400219375).
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