Chemical Engineering Journal 417 (2021) 129272

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Chemical Engineering Journal

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Fate of an antibiotic and its effects on nitrogen transformation functional

bacteria in integrated vertical flow constructed wetlands
Yucong Zheng a, b, Ying Liu a, Miaowen Qu a, Mengqing Hao a, Dan Yang a, Qian Yang a,
Xiaochang C. Wang a, c, Mawuli Dzakpasu a, c, *
a
Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE, School of Environmental and Municipal Engineering, Xi’an University of Architecture
and Technology, Xi’an 710055, PR China
b
Shaanxi Key Laboratory of Environmental Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, PR China
c
International Science & Technology Cooperation Center for Urban Alternative Water Resources Development, Xi’an University of Architecture and Technology, Xi’an
710055, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: This study aims to provide insights into the fate and effects of the antibiotic sulfamethoxazole (SMX) on nitrogen
Integrated vertical-flow constructed wetland transformation processes in integrated vertical-flow constructed wetlands (IVCW), which is useful to create
Sulfamethoxazole strategies to improve their degradation and combat antibiotic resistance dissemination. Findings indicated
Nitrogen removal
chronic exposure to SMX to cause moderate deterioration of IVCW performance for nitrogen removal (p < 0.05),
Functional bacteria
with average TN removal efficiency reductions of circa 10%. High-throughput sequencing and network analysis
demonstrated chronic exposure of antibiotics to impact functional bacteria, ammonium oxidizing bacteria (AOB)
and ANAMMOX, significantly. ANNAMMOX emerged as a potential pathway of nitrogen removal in the IVCW,
albeit with considerable accumulation of NO−3 -N (12.76 mg/L) and carbon deficiency (C/N = 0.99). Nonetheless,
IVCW provides a promising technology for the advanced treatment of antibiotic-containing wastewater,
demonstrated by a high and stable removal efficiency (circa 95%) of sulfamethoxazole. Biodegradation, pri­
marily in the first stage of the IVCW, represented the major removal pathway of SMX. Microbial network analysis
and the enrichment of Pseudomonas, Bradyrhizobium, Sphingomonas and Luteimonas demonstrated potential for
the biodegradation of SMX. These results provide a theoretical reference for the enhancement of nitrogen and
antibiotics removal in constructed wetlands.

1. Introduction compounds in wastewater, sewage sludge, and manure are conveyed to

Antibiotics are immensely beneficial to human and veterinary
healthcare. Global antibiotic consumption is increasing considerably
due to its frequent use for a variety of therapeutic and prophylactic
purposes [1,2]. Antibiotic production is presently in excess of 24,800
tons per year in China, with a growing proportion of these antibiotics
administered to poultry and livestock [2]. As antibiotic usage rises, so
too does the risk of antibiotic contamination of the environment. Anti­
biotics are only partially metabolized by humans and animals, with large
amounts of administered dosages excreted to environmental compart­
ments. Antibiotics are also directly released into wastewater from the
pharmaceutical industry and are poorly removed by wastewater treat­
ment plants (WWTPs) [1,3,4]. Consequently, active antibiotic
terrestrial and aquatic ecosystems.
A large number of antibiotics are detected in the environment at
concentrations ranging from 3.4 ng/L to 4.82 μg/L (water) or 100 ng/kg
to 5070 μg/kg (soil and sediment) [1,5–7]. These concentrations,
generally, may not be considered therapeutic and are well below the
minimum inhibitory concentrations established by acute toxicity tests.
Sub-lethal or therapeutic doses, however, can promote the development
of antibiotic resistance in both target and non-target organisms [8] and
have been found to affect the structure and function of ecologically
important microbial communities [9]. Hence, cost-efficient removal of
antibiotics from WWTP effluents and environmental water is important
and constructed wetlands (CW) may provide a sustainable option.
CW provide an alternative to traditional wastewater treatment

* Corresponding author at: Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE, School of Environmental and Municipal Engineering,
Xi’an University of Architecture and Technology, Xi’an 710055, PR China.
E-mail address: mawuli.dzakpasu@xauat.edu.cn (M. Dzakpasu).

https://doi.org/10.1016/j.cej.2021.129272
Received 21 December 2020; Received in revised form 4 February 2021; Accepted 3 March 2021
Available online 10 March 2021
1385-8947/© 2021 Elsevier B.V. All rights reserved.
Y. Zheng et al.
technologies. Their main advantages are low-cost, eco-friendly and good
contaminant treatment performance. Microbial communities play a
fundamental role in nutrient transformation in CW. This is particularly
evident in the transformation and removal of nitrogen (N). These N
transformation processes commonly include nitrification and denitrifi­
cation to reduce the concentration of organic and inorganic N prior to its
discharge into surface waters [10]. The latter step of the reduction
process, denitrification, reduces the eco-toxic compound nitrate (NO−3 -
N) to N2 or nitrous oxide (N2O), which are lost to the atmosphere. The
nitrogen removal pathways in CW, overall, include a complex interplay
of plants uptake, substrate adsorption and biodegradation [11]. The
dominant pathway is via microbial nitrification, denitrification and
ANAMMOX processes [10]. These processes require distinct oxida­
tion–reduction (redox) conditions to incubate functional bacteria, such
as aerobic nitrifiers and anaerobic denitrifiers. However, the ideal aer­
obic and anaerobic zones are unable to exist simultaneously in single-
stage CW [12]. For example, vertical down-flow CW with unsaturated
beds facilitate nitrification while vertical up-flow with saturated beds
favor denitrification [13]. Therefore, one-stage CW have limited ability
in TN removal. The flow type also influences the degradation efficiency
of antibiotics, whereby down-flow vertical CW are noted to be advan­
tageous in the removal of antibiotics [14,15]. Flow types impress a
dominant influence on the physicochemical properties such as redox
potential, consequently, impacting the distribution of specific microbial
biomass that might play important roles in antibiotic removal in CW
[14]. Thus, theoretically, the ammonium oxidation rate and redox
conditions are two critical factors impacting the N transformation and
micropollutants removal in CW. However, it is still unclear how the
functional microorganisms in CW systems would respond to the chronic
exposure of antibiotics at the molecular level, as well as the impacts of
antibiotics on the microbial community structure and reactor
performance.
Considerable advances have been made in CW design and opera­
tional processes to promote high N transformation and removal effi­
ciency. The implementation of integrated vertical-flow constructed
wetlands (IVCW) provide one such advancement. IVCW consist of a
hybrid down-flow chamber and an up-flow chamber, giving rise to
“aerobic/anoxic/anaerobic/anoxic/aerobic” multi-oxygen conditions
along the flow direction. Thus, the unique structure of IVCW could
enhance nitrogen transformation processes and, thus, nitrogen removal.
As antibiotics are introduced to influent wastewaters, however, the
resulting impact on microbial activity and N transformation may reduce
the efficacy of these efforts. Although IVCW have been applied to
remove nitrogen from various types of wastewater [16–19], only very
few studies have explored their potential for the biodegradation of an­
tibiotics with contradictory results so far [20,21]. Therefore, the
mechanisms for the removal of these chemicals need to be fully eluci­
dated in order to optimize the design criteria and performance of CW for
their removal. A better understanding of the fate and effects of antibi­
otics on CW processes is useful to create strategies to improve their
degradation and combat antibiotic resistance dissemination.
This paper aims to (i) validate the feasibility of IVCW towards
simultaneous removal of nitrogen and antibiotics, and (ii) investigate
the responses of functional bacteria (AOB and anammox) to chronic
exposure of antibiotics. Sulfamethoxazole (SMX) is selected as a typical
antibiotic for the experiment. SMX is a broad-spectrum antibiotic
belonging to the class sulfonamide. It is frequently detected in the water
environment due to its chemical stability and hydrophilicity [14]. The
average concentration of SMX in natural surface water ranges from
67.85 to 2231.0 ng/L [22]. In-depth analysis of the shifts in functional
bacteria involved in nitrogen transformation and SMX degradation via
the analysis of microbial networks provides a theoretical reference for
the enhancement of nitrogen and antibiotics removal in CW.
2
Chemical Engineering Journal 417 (2021) 129272
2. Materials and methods
2.1. Experimental setup and operation
Two sets of lab-scale IVCW were established in parallel, of which one
was fed with wastewater containing sulfamethoxazole (IVCW-S) and the
other served as control experiment without sulfamethoxazole stress
(IVCW-C) (Fig. 1). Each set of IVCW consists of two polymethyl meth­
acrylate columns (inner diameter of 15 cm and height of 100 cm)
wrapped with aluminum foil. The first stages of the IVCW, DC and DS,
were unsaturated with down-flow while the second stages, UC and US,
were saturated with up-flow. The substrate comprised a 55 cm-layer of
gravel (Φ2-6 mm) on the top and a 25 cm supporting layer of coarse
gravel (Φ10-20 mm) at the bottom. It has been reported that the pres­
ence of vegetation can promote the removal efficiency of CW treating
antibiotic-abundant wastewater [20,23] via direct plant uptake and
enhancement of rhizosphere microbial degradation. Phragmites australis,
a widely used plant in CW, was cultivated in this study at an initial
density of 4–5 stalks per column. Previous studies also observed that
Phragmites australis showed tolerance to antibiotics at the concentrations
ranging from 0.1 μg/L to 1000 μg/L [24].
The reactors were operated continuously at a hydraulic loading rate
of 0.2 m3/(m2⋅d) and hydraulic retention of 2 d. The influent was freshly
prepared every two days with tap water and nutrients, including
glucose, NH4Cl, KNO3 and KH2PO4 to simulate domestic sewage. The
average influent concentrations of COD, NH+ 4 -N, NO3 -N, TN and TP
−
were 559.79, 20.63, 10.59, 32.34 and 4.31 mg/L, respectively. The
influent SMX concentration was kept at 100 μg/L by adding the stock
solution. The concentration of SMX tested was shown by previous re­
searchers to be suitable for assessing CW antibiotic removal potential
[25]. Both setups, IVCW-S and IVCW-C, were operated under identical
conditions with ambient temperature of circa 25℃ and exposure to
artificial sunlight to simulate field conditions over 12 months from
January to December in 2018.
2.2. Water sampling and analysis
Waters samples were collected from the influent and effluent of the
first and second stages of both IVCW-S and IVCW-C weekly. Water
quality parameters, including COD, TN, NH+4 -N, NO3 -N and NO2 -N were
− −
analyzed according to standard methods [26] (MEPC (Ministry of
Environmental Protection of China), WWMAA, (Water, Wastewater
Monitoring), 2002). The removal efficiencies and rates were calculated
as follows:
(Cin ⋅Vin - Cout ⋅Vout )
Removal efficiency (%) = × 100 (1)
Cin ⋅Vin
Removal rate (g⋅m - 2 ⋅d - 1 ) = q × (Cin - Cout ) (2)
where Cin is the influent concentrations (mg/L); Cout is the effluent
concentrations (mg/L); Vin and Vout are corresponding influent and
effluent volumetric flow rate (m3/d); q is the hydraulic loading rate
(HLR, m3/(m2⋅d)).
The analysis of the removal effect of SMX was based on dynamics of
SMX concentration in the first and second stages of IVCW-S. The sam­
pling frequency was kept at every two days during the operation period
except February for that sampling activities were not carried out in
February. Ultra-high performance liquid chromatography tandem mass
spectrometry (UPLC-MS/MS) (Acquity TQD, Waters, USA) was
employed to measure concentrations of SMX in water samples prefil­
tered with 0.22 μm filters. Chromatographic separation was performed
by using a reversed phase ACQUITY BEH C18 column (2.1 mm × 100
mm; 1.7 μm) (Waters, USA) with the temperature set to 35℃. The
temperature of the sample manager was set to 25℃. The mobile phases,
delivered by a binary pump, consisted of water (A phase) and
Y. Zheng et al.
Fig. 1. Schematic diagram of the integrated vertical flow constructed wetland experimental setups.
acetonitrile (B phase). Flow rate was set to 0.2 mL/min and the injection
volume was 10 μL. The total run time of the chromatographic analysis
was 7.5 min. Gradient elution was used for optimum separation, with
the gradient set up as follows: 45% B phase (0–1 min), 52% B phase (1–4
min), 55% B phase (4–7.5 min). Tandem mass spectrometry detector
with ESI source was operated in positive ionization mode. Parameters of
the mass spectrometry are presented in Table S1. The limit of quantifi­
cation (LOQ) was 20 ng/L, defined as ten times the ratio of signal to
noise.
2.3. Substrate sampling and microbial community analysis
Substrate samples were collected at different depths from each stage
of the IVCW at sampling points labelled C#1, C#2, C#3, C#4, C#5,
C#6, S#1, S#2, S#3, S#4, S#5 and S#6 (Fig. 1). Particles attached onto
the substrate were rinsed using ultra-pure water and collected into a 50
mL centrifuge tube, which were then centrifuged at 9000 rpm for 10
min. Samples collected from the same column were homogenized,
consequently resulting in a total of four substrate samples (DC, UC, DS
and US) for microbial analysis. The V4-V5 regions of bacterial 16S rRNA
were selected as taxonomy specific fragments. The samples were stored
at − 80℃ before being sent over to Sangong BioTech, Inc. (Shanghai,
China) for DNA extraction and Illumina MiSeq sequencing analysis.
2.4. Statistical analysis
SPSS 25 package (IBM SPSS Inc. Chicago, USA) was used for statis­
tical data analysis. PCA was performed using CANOCO 5.0. Spearman
correlations between physicochemical parameters and dominant genera
were obtained by using R Statistical Software (v.3.6.2; http://www.
r-project.org/). Strong (|R| > 0.6) and significant (p < 0.05) correla­
tions were used to visualize the correlation between the microbial
composition and the physicochemical parameters by network analysis.
Gephi (v.0.9.2; The Gephi Consortium, Paris, France) was employed to
carry out network analysis.
3
Chemical Engineering Journal 417 (2021) 129272
3. Results and discussions
3.1. SMX removal in IVCW
Concentration dynamics of SMX along the flow direction and the
total removal efficiencies are shown in Fig. 2. Influent concentrations of
SMX varied appreciably, ranging between 76.30 and 115.58 µg/L.
Similarly, the removal efficiency of SMX fluctuated greatly in the first
three months of operation. However, the removal efficiency stabilized as
the wetlands matured, akin to microbial biomass enrichment and bio­
film formation, which made the system more conductive to SMX
removal [27]. The average removal efficiency of SMX reached 95.40%,
with a maximum of 98.33%, thereafter. The first stage (DS) accounted
for a large proportion of the attenuation of SMX, with an average
contribution of 95.36% to the total removal. Significant positive corre­
lations between the removal rates of COD and SMX in DS (R = 0.34, p <
0.05) (Fig. S1) indicated SMX could be more readily removed when co-
existing with other readily biodegradable carbon sources [28–30].
Organic substrate is demonstrably crucial for the catalysis of antibiotic
degradation [31]. Because of sufficient oxygen and carbon source sup­
ply, the first stages, down-flow columns, are more conductive to the
proliferation of aerobic heterotrophic bacteria contributing to the
attenuation of recalcitrant organic matter. Therefore, the operation
mode contributed to the impressive SMX removal effect of the first stage
of the IVCW used in this study. Nonetheless, the second column (US) of
the IVCW used in this study also showed appreciable removals of the
residual SMX, with an average removal efficiency of 36%. Furthermore,
the SMX concentration of US ranged along with that of DS (Fig. 1). The
removal efficiency of SMX in US decreased as the concentration levels
increased in DS. Up-flow CW with longer HRT could promote the
adsorption of antibiotics [20,32,33]. Existing at relatively lower con­
centrations, SMX appeared to be adsorbed instead of biodegraded in US,
albeit the initial concentration of SMX has an important effect on this
removal process [32]. Biodegradation, adsorption and plants absorption
are reportedly responsible for antibiotics removal in CW [34]. Among
these, plants absorption was demonstrated to contribute<1% of total
removal [20,35]. Therefore, based on the above discussion, biodegra­
dation and substrate adsorption are deemed to constitute the main
removal mechanism in IVCWs.
Y. Zheng et al. Chemical Engineering Journal 417 (2021) 129272

Fig. 2. Removal efficiency of sulfamethoxazole in integrated vertical flow constructed wetlands.

The performance of IVCWs in this study exceed that of other types of stage CW.
IVCWs used for sulfonamides removal. In former studies, researchers
employed an IVCW composed of special substrate materials such as
3.2. Nitrogen removal in IVCW
clinoptilolite, zeolite, and quartz sand to treat swine wastewater
enriched with antibiotics [20]. The results showed removal efficiencies
The concentrations of the influent and effluent parameters from the
for three sulfonamides of only 56.1%-68.8%. This finding demonstrate
IVCWs during the operation and the calculated removal efficiencies are
biodegradation, primarily in the first stages, to represent the dominant
listed in Table 1. The average concentrations of TN in the effluents of
removal pathway of SMX in IVCWs. Similarly, it was observed that
IVCW-C and IVCW-S were 11.65 mg/L and 14.80 mg/L. The corre­
biodegradation contributed over 60% of the total SMX removal while
sponding average removal efficiencies of TN were 63.98% and 54.24%
adsorption only contributed about 20% in the removal experiments
at the average influent concentration of 32.34 mg/L for IVCW-C and
using zeolite as substrate and the aerobic condition could promote the
IVCW-S, respectively, comparable to previous studies employing IVCW
removal efficiency [36]. Previous studies also showed that the removal
to treat wastewater (Table S2). Distinct transformation processes of ni­
efficiency of SM2 in down-flow vertical constructed wetlands could
trogen in the first and second stages contributed to the removal of TN.
reach 68–73%, attributable to system soil sorption [37]. However, sul­
4 -N concentrations were reduced to 2.57 mg/L
In the first stages, NH+
fonamide antibiotics with high solubility exhibit a low sorption ability in
(DC) and 2.91 mg/L (DS). Nonetheless, stable removals of NH+ 4 -N were
soil, which may limit the removal effect of the system [11]. In contrast,
achieved in both DC and DS with average removal efficiencies of 87.54%
fluoroquinolones and tetracyclines with high soil–water distribution
and 85.89%. However, the concentration of NO−3 -N exhibited an oppo­
coefficient (log Kd) are mainly removed via substrate adsorption in CW.
site trend with the average effluent concentrations of the first stages
Song et al. [32] observed that the removal efficiency of tetracycline
(12.66 mg/L in DC and 12.76 mg/L in DS), exceeding the influent
reached 98% in up-flow vertical CW and its adsorption to wetland
(10.59 mg/L). This observation indicated that the produced NO−3 -N via
substrate seemed to be promoted by the relatively high log Kd value.
nitrification process was not well denitrified. Therefore, the first stages
Overall, due to the multiple pollutant removal pathways, such as
might be dominated by nitrification process and the increase of nitrate
biodegradation and substrate adsorption, IVCWs might be more
concentrations revealed limited microbial capacity of denitrifiers in
conducive to the efficient and stable removal of antibiotics than single
unsaturated substrate under the down-flow operation mode, consistent

Table 1
Characteristics of influent and effluents in each column.
Parameters Influent (mg/L) Effluent (mg/L) Average removal efficiency (%)

DC UC DS US DC UC IVCW-CTotal RE DS US IVCW-STotal RE

COD 559.79 21.57 15.29 21.30 14.76 96.15** 29.11** 97.27 96.20** 30.70** 97.36
(80.96) (14.76) (12.79) (8.82) (9.27)
TN 32.34 17.53 11.65* 19.39 14.80* 45.79** 33.54** 63.98* 39.92** 23.67** 54.24*
(2.29) (4.86) (3.65) (4.78) (4.37)
NH₄⁺ -N 20.63 2.57 0.27* 2.91 1.76* 87.54 89.49 98.69* 85.89 39.52 91.67*
(2.19) (1.11) (0.57) (2.60) (1.41)
NO₃ -N
−
10.59 12.66 9.25* 12.76 11.16* − 19.55** 26.94** 12.65* − 20.49** 12.54** − 5.38*
(1.04) (3.09) (3.81) (2.62) (4.52)

All the data is presented as mean (standard deviation) (n = 37).

RE (Removal Efficiency).
*Significant differences between IVCW-C and IVCW-S (p < 0.05).
**Significant differences between the first and second stages of IVCW (p < 0.05).

4
Y. Zheng et al.
with the results of previous studies [11,19,38]. Waterdrop influent was
also proved to be a feasible way to strengthen the oxygen supplemen­
tation and nitrification process in CW [12]. Moreover, though the
biodegradation of SMX might consume oxygen and substrates involved
in nitrifiers metabolism, the removal efficiency of NH+ 4 -N was not
significantly affected in DS compared to DC (p > 0.05). This was likely a
result of the aerobic condition created by the operation mode, which
made the first stages ideal aerobic units with an effective nitrification
process.
Despite the saturated condition, the second stages also served well
for supplemental attenuation of NH+ 4 -N. The low DO of 1.96 and 2.96 for
UC and US might favor denitrification process as well [38]. Therefore,
both nitrification and denitrification processes might occur in the second
stages with the effluent concentration of NH+ 4 -N and NO3 -N decreasing
−
simultaneously. Notably, a significant positive correlation was observed
between the removal rates of NH+ 4 -N and NO2 -N in US (R = 0.36, p <
−
0.05) (Fig. S1). Previous studies reported that under oxygen limited
conditions, ANAMMOX could be the alternative nitrogen removal pro­
cess, which could also contribute to the removal of NH+ 4 -N, especially
coexisting with NO−2 -N [19,39]. Therefore, it is speculated that simul­
taneous nitrification, ANAMMOX and denitrification process (SNAD)
might take place in US under SMX stress. In this process, NH+ 4 -N was
partially oxidized into NO−2 -N and then the remaining NH+ 4 -N was
oxidized with the produced NO−2 -N as electron acceptors by ANAMMOX
bacteria. Subsequently, the byproduct, NO−3 -N of the ANAMMOX pro­
cess was reduced by denitrifying bacteria [10]. However, based on the
average removal efficiency of 26.94% (UC) and 12.54% (US), NO−3 -N
was not well denitrified due to the lack of carbon source (C/N =
0.99–1.31), with majority of organic matter being removed in the first
stages. Although glucose could provide electrons for denitrification,
COD concentrations were reduced by 29.11% and 30.70% in the second
stages (Table 1). It is noted that the ratio of nitrate/COD is 1:1.74 in
denitrification, and denitrifying microorganisms could only exert a
sufficient reduction ability when C/N ratios are over 10 [10,40]. Carbon
source is a defining factor in the process of nitrogen removal since it is
vital for the growth of heterotrophic bacteria, such as denitrifiers [31].
Carbon deficiency would cause competition between NO−3 -N and NO−2 -N
for electrons, resulting in low denitrification rates and, consequently,
leading to the limited utilization of organic matter. In the present study,
NO−2 -N accumulation was observed in the second stages (0.20–0.28 mg/
L) with the average concentration 7–9 times higher than that in the first
stages (0.03 mg/L). The result of a former study signified that abundant
carbon addition was necessary to prevent NO−2 -N accumulation in the
denitrification process [39].
Consequently, the removal efficiency of nutrients, except NH+ 4 -N,
significantly differed between the first and second stages (p < 0.05),
caused by the distinct functional processes occurring in the two stages
under different hydraulic conditions (Table 1). Furthermore, long-term
exposure to SMX appears to appreciably inhibited the nitrogen
removal in IVCW-S, based on the analysis of effluent nutrient concen­
trations of the second stages, UC and US (p < 0.05), consistent with
former studies [20,41]. However, there was no significant difference in
the effluent nutrient concentrations of the first stages, DC and DS (p >
0.05). A possible explanation is that the nitrogen removal pathways
might transform due to the effect of SMX in IVCW-S. In other words, the
introduction of SMX might potentially drive the shift of microbial
Table 2
Microbial richness and diversity estimation in each column of the IVCW.
Sample_ID OTU_num Richness estimator
ACE Chao1
DC 613 711.28 682.24
UC 1744 2230.74 2149.59
DS 950 1185.05 1152.90
US 1868 2425.73 2435.24
5
Chemical Engineering Journal 417 (2021) 129272
communities and certain ecological functions for nitrogen cycling.
Further discussion is presented in sections 3.3 and 3.4.
3.3. Shifts in microorganism community composition by SMX
Microbial communities in the substrate samples of IVCW-C and
IVCW-S were analyzed using high-throughput sequencing. At 97% of
similarity level, the OTUs abundance of each sample fluctuated within
the range of 613–1868, with coverage ranging from 98.62% to 99.80%
(Table 2). These results signify that the sequencing depth included the
majority of microbial species in all samples. The richness estimators and
diversity indexes of the microbial community are shown in Table 2.
Some particular trends were observed to highlight the influence of SMX
and hybrid hydraulic conditions on bacterial diversity in the substrate
samples. Microbial richness, estimated by Chao1 and ACE, was higher in
IVCW-S than that in IVCW-C. Nonetheless, the diversity indexes showed
the same tendency. The increase in richness and diversity of the mi­
crobial communities in IVCW-S may be attributed to the introduction of
antibiotics stimulating the growth of some microbial species [42]. At
environmental concentrations, antibiotics can serve as signaling mole­
cules and stimulate the metabolic activity of bacteria [31]. In addition,
the microbial richness and diversity recorded for the second stages were
significantly higher than those of the first stages in both IVCW-C and
IVCW-S. The possible reason for this phenomenon is that the microor­
ganisms that function in the removal process were retained as dominant
species while other microorganisms were eliminated, with majority of
pollutants removed in the first stages. Moreover, it was also proved that
saturated substrate could contribute to the proliferation of microbial
species with larger attachment sites for biofilm formation [43].
PCA analysis was performed to evaluate the difference in microbial
communities’ structures of IVCW-C and IVCW-S. The first and second
axis explained 69.24% and 23.82% of the total variation in the bacterial
community, respectively (Fig. 3a). The substrate samples in the second
stages were clustered in the same group apart from the first stages due to
the different oxygen condition and function. However, the bacterial
community structure differed significantly in the first stages, indicating
that the addition of SMX significantly transformed the microbial com­
munity structure in DS. It has been reported that microorganisms are
highly sensitive to exogenous contaminants, including antibiotics, and
can be responsible for antibiotic removal by using antibiotics as carbon
sources [27,44]. Therefore, despite the lower diversity, the first stages
might be abundant with bacteria responsible for pollutants removal,
demonstrating the relatively high removal performance of the first
stages for nutrients and SMX.
Likewise, the results of high-throughput sequencing showed that
there were significant differences in microorganism composition at both
phyla and genera levels due to different hydraulic conditions and the
stress of SMX. At the phylum level, the phyla with the top ten relative
abundances, accounting for >97% of the total abundance, were selected
for analysis. The most dominant phylum of each sample was Proteo­
bacteria (46.95%-72.93%), followed by Planctomycetes (6.17%-
24.79%) and Bacterodetes (5.01%-11.90%) (Fig. 3b), which were
widely distributed and detected with high frequency in wetlands treat­
ing antibiotics-containing wastewater [27,45]. Due to its hydraulic
conditions and role in nutrients removal, Proteobacteria and Bacter­
odetes were more likely to be abundant in the first stages while
Coverage (%) Diversity index
Simpson Shannon
99.80 0.0815 3.88
99.17 0.0141 5.34
99.55 0.0271 4.66
98.62 0.0127 5.68
Y. Zheng et al.
Fig. 3. Microbial communities in IVCWs based on the results of high-throughput sequencing: (a) Principal Component Analysis (PCA) of each column at OTU level;
relative abundance of bacteria at (b) phylum level; (c) genus level.
Planctomycetes exhibited the opposite trend, being more abundant in
the second stages (Fig. 3b). Proteobacteria are integral to the removal of
nutrients and some organic pollutants [11,46]. Bacteroidetes can pro­
liferate well in wastewater with high organic matter content and ac­
count for protein degradation [46]. The higher abundance of
Proteobacteria and Bacteroidetes in the first stages may conduce to the
remarkable removal performance for nutrients. Planctomycetes function
in ANAMMOX widely, which is commonly observed in wetlands but not
frequently detected in the constructed wetlands treating antibiotics. It
has been suggested that the relative abundance of these phyla was
notably affected by the presence of the antibiotics [47]. However, in the
present study, the addition of SMX appears to promote the growth of
Proteobacteria and Firmicutes in IVCW-S, which corroborates the find­
ings of Man et al. [45]. Of these two phyla, Proteobacteria was reported
to include a variety of antibiotic-resistant bacteria (ARB) [43]. Mean­
while, Planctomycetes were enriched in the IVCW-S, especially in US,
with a relative abundance of 24.79%, far higher than that of UC
(10.1%). Dedysh and Ivanova [48] reported Planctomycetes to be most
abundant at the aerobic/anoxic interface of wetlands. In order to ach­
ieve the ideal ANAMMOX process, former studies have stated that aer­
obic and anaerobic conditions are required for partial nitrification
(NH+ 4 -N → NO2 -N) and ANAMMOX (NH4 -N + NO2 -N → N2), respec­
− + −
tively [31]. Therefore, the complex “aerobic/anoxic/anaerobic/anoxic/
aerobic” microenvironment along the water flow direction of the IVCW
may contribute to the proliferation of Planctomycetes. Also, the
biodegradation progress of SMX may amplify this effect, which, conse­
quently, influences the nitrogen transformation pathways in US.
6
Chemical Engineering Journal 417 (2021) 129272
At the level of genera, the top 50 genera, accounting for >84% of the
total abundance, were selected for analysis. The dominant genera of
both IVCWs mainly belonged to Proteobacteria and Planctomycetes
(Fig. 3c). However, due to the functional difference, the relative abun­
dance of the genera differed considerably in the two stages. In both
IVCW-C and IVCW-S, the relative abundance of nitrifying bacteria,
Rhizomicrobium, Rudaea, Mizugakiibacter, and bacteria related to organic
carbon degradation, Reyranella and Sphingorhabdus, were significantly
higher in the first stages than that in the second stages. Furthermore, the
relative abundance of denitrification bacteria such as Pseudolabrys,
Altererythrobacter, Rhodobacter and Raoultell exhibited an opposite trend,
being concentrated in the second stages (Fig. 3c). The difference in
distribution of these genera might be attributed to the different capac­
ities of down-flow and up-flow vertical constructed wetlands for
ammonium oxidation and denitrification [38]. More importantly, the
introduction of SMX appears to transform the microbial composition
involved in nitrogen cycling in IVCW-S. Specifically, the relative abun­
dance of nitrifying bacteria, Rhizomicrobium (DS: 1.87%; DC: 6.3%) and
Rudaea (DS: 0.01%; DC: 1.13%) were significantly lower in DS. This
finding corroborates reports that the activity of ammonium-oxidizing
bacteria (AOB) may be inhibited in the presence of pharmaceutical
and personal care products [49]. Moreover, it is worth noting that
Zavarzinella, Pirellula, Blastopirellula and Gemmata were enriched in US
with relative abundances higher than UC by 1.73%, 3.08%, 3.54% and
1.32%, respectively. These four genera were proved to be ANAMMOX
functional bacteria belonging to Planctomycetes [50]. Moreover,
compared with UC, the relative abundance of denitrification bacteria,
Y. Zheng et al.
Rhodobacter, Pseudolabrys, Altererythrobacter and Raoultella in US
decreased by 1.03%, 1.16%, 2.46%, and 2.32%, respectively, which
might result from the substrate competition over NO−2 -N for ANAMMOX
and denitrification [39]. In addition, the biodegradation process of SMX
exerted selective pressure on the community structure. Luteimonas was a
unique bacterium in IVCW-S with a remarkably high relative abundance
of 9.94%. Reports indicate that Luteimonas could be involved in the
degradation of ampicillin [51]. Here, it is also speculated that Luteimo­
nas could be involved in the degradation of SMX. However, further ev­
idence to support this claim should be pursued in future studies.
Specifically, further investigation is required to seek the antibiotic-
resistance mechanism supporting the substantial proliferation of Lutei­
monas in IVCW under SMX stress. The organic carbon-degrading bac­
teria, Reyranella and Sphingorhabdus were abundant in DS with the
relative abundances of 2.73% and 1.46% compared with that of 1.43%
and 0% in DC, respectively. Compared to the other columns (DC, UC and
US), DS was more abundant with Pseudomonas (2.18%), Bradyrhizobium
(1.16%) and Sphingomonas (1.71%), which are frequently reported as
ARB [23,43]. Moreover, the abundance of other bacteria following the
top 50 genera, marked as Others in Fig. 3c, increased in both DS and US
compared to DC and UC. This observation indicates that the introduction
of SMX stimulated the growth of some microbial species, consequently
increasing the diversity and richness of IVCW-S, consistent with the
results obtained at the OTU level.
3.4. Correlation of microbial composition and the removal of SMX and
nitrogen
Nitrogen transformation processes, namely nitrification, denitrifi­
cation and ANAMMOX, are driven by microorganisms in CW. To achieve
optimal removal efficiency, a variety of functional bacteria are involved
in the transformation process of nitrogen [10]. Sulfonamides removal
pathways via microorganisms are also considered the main mechanisms
in CW [28]. Based on the results of microbial community and discussion
in the above sections, nitrogen removal processes and the relative
abundance of bacteria involved appear to have shifted due to the
introduction of SMX in IVCW-S. Network analysis was used to explore
the removal pathways of nitrogen and SMX in IVCW-C and IVCW-S and
Fig. 4. Network analysis showing correlation between removal rates of pollutants and bacterial communities based on top 50 genera except Unclassified genus. The
nodes are colored according to modularity class. Red lines represent significantly strong (R > 0.6, p < 0.05) positive linear relationships, blue lines represent strong
(R < − 0.6, p < 0.05) negative linear relationship.
7
Chemical Engineering Journal 417 (2021) 129272
its correlations with the microorganism composition. The network
comprising of TN, NH+ 4 -N, NO3 -N, NO2 -N, SMX and the top 50 genera
− −
(except Unclassified) is shown in Fig. 4. The network for IVCW-C consists
of 51 nodes and 460 edges (Fig. 4a) while that for IVCW-S consists of 54
nodes and 388 edges (Fig. 4b). The associations between genera
decrease significantly in IVCW-S compared to IVCW-C, suggesting that
functional division and identification of bacteria is far clearer in IVCW-
S. Both of the networks exhibit modular structures comprising four
major modules (Fig. 4). However, the modules are driven by different
parameters and dominated by different genera due to the introduction of
SMX.
The removal rates of NH+ 4 -N and COD are gathered in the same
Module, Module C1 in IVCW-C (Fig. 4a). Module C1 is dominated by the
genera of nitrifiers, Rhizomicrobium and Rudaea, both of which are
positively related to the removal of NH+ 4 -N and COD. However, the
removal rate of NH+ 4 -N is clustered with NO2 -N in IVCW-S. The removal
−
rate of NH4 -N is widely related to the genera Pirellula, Blastopirellula and
+
Gemmata that function in the ANAMMOX process instead of nitrifiers in
IVCW-S (Fig. 4b). Therefore, SNAD process might be the dominant
pathway contributing to NH+ 4 -N removal under SMX stress in the second
stage (US) based on the high-throught sequencing results. However, the
reaction between NH+ 4 -N and NO2 -N, producing N2 and NO3 -N might be
− −
inhibited with the influent of US being NO−3 -N-dominated [39]. In
addition, the nitrifiers clustered in Module C1 with NH+ 4 -N, namely,
Rhizomicrobium and Rudaea, are also clustered in Module S4 apart from
the removal of nitrogen in IVCW-S. Conversely, the nitrfiers, Miz­
ugakiibacter and Burkholderia are stimulated to function in NH+ 4 -N
removal process (Fig. 4b). These findings explain why the effluent
concentrations of NH+ 4 -N were higher in IVCW-S than that in IVCW-C.
Furthermore, the removal rate of COD is clustered in Module S1
together with that of SMX in IVCW-S (Fig. 4b). Module S1 is dominated
by the genera Luteimonas, Pseudomonas, Bradyrhizobium, Sphingomonas,
Reyranella, and Sphingorhabdus. Among these genera, the relative
abundance of Pseudomonas, Reyranella, and Sphingorhabdus positively
correlated with the removal rates of both SMX and COD while those of
Luteimonas, Bradyrhizobium, and Sphingomonas correlated to SMX posi­
tively. Some bacteria can assimilate simple external carbon sources to
induce nonspecific enzymes for the degradation of recalcitrant organics
Y. Zheng et al.
via co-metabolism [30]. Pseudomonas, Reyranella and Sphingorhabdus
might utilize glucose to metabolize and, subsequently, attenuate SMX
via the co-metabolism of organic matter and SMX in that these three
genera are not linked to the removal rate of COD in IVCW-C (Fig. 4a).
Moreover, the differential abundance of ARB, Pseudomonas, Bradyrhi­
zobium, and Sphingomonas could be potential bacterial biomarkers and
served as functional bacteria during the removal process of SMX in
IVCW-S [52]. According to previous research, Pseudomonas is one of the
main functional bacteria participating in the biodegradation of SMX
[28,30]. Therefore, the genera having a similar ecological niche with
Pseudomonas might be potential degrading bacteria for SMX, especially
Luteimonas, the unique bacteria in IVCW-S, primarily the first stage.
The dominant genera involved in denitrification, Pseudolabrys,
Altererythrobacter, and Raoultella, are clustered in Module C4 related to
the removal rate of NO−3 -N in IVCW-C (Fig. 4a). All of the three genera
are abundant in UC, the second stage of IVCW-C and positively corre­
lated with the removal rate of NO−3 -N. This finding indicates that the
saturated substrate in the second stages promote the proliferation of
denitrifiers and the denitrification process. Therefore, insufficient car­
bon source could be the predominant reason for the limited removal of
NO−3 -N in IVCW-C. However, only Pseudolabrys and Raoultell positively
correlated with the removal rate of NO−3 -N in IVCW-S (Fig. 4b). As dis­
cussed in section 3.3, the relative abundance of denitrifiers decreased in
the second stage of IVCW-S compared to the corresponding stage of
IVCW-C. Among the four dominant genera, Rhodobacter, Pseudolabrys,
Altererythrobacter and Raoultella, Pseudolabrys and Raoultell still serve as
the functional bacteria for denitrification. Conversely, the other genera
with similar niches might not be involved in nitrate removal in IVCW-S,
which might contribute to the poor NO−3 -N removal performance of
IVCW-S.
Overall, by altering the composition of the microorganisms associ­
ated with the removal of nitrogen, the introduction of SMX altered the
nitrogen removal processes and, consequently, affected the performance
of IVCW-S in the attenuation of nitrogen adversely.
3.5. Proposed removal mechanisms of nitrogen and SMX in IVCW
Based on the discussion in the sections above, we elucidate the
Fig. 5. Possible microbial nitrogen and antibiotic (SMX) removal pathways and corresponding functional bacteria in IVCW. The values in the charts were calculated
based on the average concentration of nitrogen in the two columns of the IVCW (Table 1).
8
Chemical Engineering Journal 417 (2021) 129272
microbial nitrogen and antibiotic (SMX) removal pathways and corre­
sponding functional bacteria (Fig. 5). Aerobic degradation and co-
metabolism via microbes constituted the main removal mechanisms of
SMX in the IVCW. Although the introduction of SMX caused a slight
deterioration of nitrogen removal performance, IVCW are feasible for
co-removal of antibiotics and nitrogen, to a large extent. The dominant
removal pathways of nitrogen under antibiotic-stress were determined
using the average concentrations (Table 1) and the following stoichio­
metric relationships in the nitrogen cycling process [39,53]: (i) the
molar ratio of NH+ 4 -N/NO3 -N is 1:1 in nitrification process; (ii) the
−
molar ratio of NH4 -N/NO2 -N is 1:1.32 with 0.26 mol NO−3 -N produced
+ −
in ANAMMOX process; (iii) 3.704 mg/L of COD is required to remove 1
mg/L of NO−3 -N in denitrification process.
Nitrification-denitrification process was dominant in the first stage,
DS of the IVCW, contributing 15.55 mg/L of N removal. SNAD process
contributed 2.58 mg/L of N removal in the second column, US of the
IVCW. The consumed COD was 6.33 mg/L, accounting for 96.86% of the
total removal (6.54 mg/L) in US of the IVCW. Thus, SNAD might be a
potential nitrogen removal process in IVCWs under antibiotic stress, but
nitrification–denitrification still serve as the dominant mechanisms of
nitrogen removal. Considering the impressive performance of the first
column of the IVCW for nitrification, and the moderate accumulation of
nitrates (12.76 mg/L) and carbon deficiency (C/N = 0.99) in the second
column, introduction of a carbon source to the second column might
present a viable approach to enhancing the total nitrogen removal
performance of IVCWs. The second column is operated under saturated
conditions, and so anaerobic/anoxic conditions prevail. This concept
has been verified in our previous study, whereby the denitrification
performance could be increased by circa 30% via the addition of
alkaline-heated cattails as carbon resource [54].
4. Conclusions
IVCW is a promising technology for the advanced treatment of
antibiotics-containing wastewater, demonstrated by a high and stable
removal efficiency (circa 95%) of sulfamethoxazole. Biodegradation,
primarily in the first stage, represented the major removal pathway of
antibiotics in IVCW. The first stage, with unsaturated substrate, create
Y. Zheng et al.
ideal aerobic conditions, conductive to the proliferation of nitrifiers and
the enrichment of Pseudomonas, Bradyrhizobium, Sphingomonas and
Luteimonas for the biodegradation of antibiotics. The second stage, with
saturated substrate, which usually favor denitrification (without anti­
biotic addition) appears to favor ANAMMOX (with antibiotic addition)
and corresponding functional bacteria. Nonetheless, the findings indi­
cated chronic exposure to antibiotics to cause significant deterioration
of IVCW performance for nitrogen removal (p < 0.05), with average TN
removal efficiency reductions of circa 10%. These findings provide in­
sights into optimization methods for simultaneous removal of nitrogen
and antibiotics.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China [grant number 51808431]; Fund for Postdoctoral Scientific
Research Project, China [grant number 2017M613290XB]; Scientific
Research Program Funded by the Shaanxi Provincial Education
Department, China [grant number 17JS078]; Scientific Research Foun­
dation for the PhD [Xi’an University of Architecture and Technology,
China, number 1608719031, 1608319013].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cej.2021.129272.
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