The performance and degradation mechanism of sulfamethazine from wastewater using IFAS-MBR

Article abstract of DOI:10.1016/j.cclet.2019.08.031

Sulfamethazine (SMZ) is an important sulfonamide antibiotic. Although the concentration in the environment is small, it is harmful. The drug residues can be transferred, transformed or accumulated, affecting the growth of animals and plants. In this study, the integrated fixed-film activated sludge membrane bioreactor (IFAS-MBR) were constructed to investigate the performance and degradation mechanism of SMZ. The addition of SMZ had a significant impact on the removal of the chemical oxygen demand (COD) and ammonia nitrogen (NH₄ ⁺-N). The optimal operating conditions were hydraulic retention time (HRT) at 10 h and solid retention time (SRT) at 80 d, respectively. On this basis, the effects of different SMZ concentrations on nutrient removal, degradation, and sludge characteristics were compared. The removal efficiency of SMZ increased with the increase of SMZ concentration. The maximum removal rate was as high as 87%. The SMZ dosage also had an obvious effect on sludge characteristics. As the SMZ concentration increased, the extracellular polymer substances (EPS) concentration and the membrane resistance both decreased, which were beneficial for the reduction of membrane fouling. Finally, seven kinds of SMZ biodegradation intermediates were identified, and the possible degradation pathways were speculated. The microbial community results showed that the microbial diversity and richness in the reactor decreased after adding SMZ to the influent. The relative abundance of Bacteroidetes, Actinobacteria, Saccharibacteria and Nitrospirae increased at the phylum level. Sphingobacteria and Betaproteobacteria became dominant species at the class level. The relative abundance of norank-p-Saccharibacteria and Nitrospirae increased significantly, and norank-p-Saccharibacteria may be the dominant bacteria for SMZ degradation.

Full text of DOI:10.1016/j.cclet.2019.08.031

Journal Pre-proof
The performance and degradation mechanism of sulfamethazine from
wastewater using IFAS-MBR
Huanhuan Hou, Liang Duan, Beihai Zhou, Yuan Tian, Jian Wei,
Feng Qian
PII:
S1001-8417(19)30504-2
https://doi.org/10.1016/j.cclet.2019.08.031
CCLET 5182
DOI:
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Chinese Chemical Letters
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15 August 2019
20 August 2019
Please cite this article as: Hou H, Duan L, Zhou B, Tian Y, Wei J, Qian F, The performance
and degradation mechanism of sulfamethazine from wastewater using IFAS-MBR, Chinese
Chemical Letters (2019), doi: https://doi.org/10.1016/j.cclet.2019.08.031
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Communication
The performance and degradation mechanism of sulfamethazine from wastewater
using IFAS-MBR
Huanhuan Houa,ᵇ, Liang Duanᵇ,, Beihai Zhoua,*, Yuan Tianᵇ, Jian Weiᵇ, Feng Qianᵇ
a Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, School of Energy and Environmental Engineering, University of Science and
Technology Beijing, Beijing 100083, China
b State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China
 Corresponding authors.
E-mail addresses: duanliang@craes.org.cn (L. Duan), zhou_beihai@126.com (B. Zhou).
Graphical abstract
The integrated fixed-film activated sludge membrane bioreactors (IFAS-MBR) were constructed to investigate the performance and
degradation mechanism of sulfamethazine (SMZ) from wastewater. Seven kinds of SMZ biodegradation intermediates were identified
and the possible degradation pathways were speculated.
ARTICLE INFO
ABSTRACT
Sulfamethazine (SMZ) is an important sulfonamide antibiotic. Although the concentration in the
environment is small, it is harmful. The drug residues can be transferred, transformed or accumulated, affecting
the growth of animals and plants. In this study, the integrated fixed-film activated sludge membrane bioreactor
(IFAS-MBR) were constructed to investigate the performance and degradation mechanism of SMZ. The
addition of SMZ had a significant impact on the removal of the chemical oxygen demand (COD) and ammonia
Article history:
Received
Received in revised form
Accepted
Available online
+
nitrogen (NH4 -N). The optimal operating conditions were hydraulic retention time (HRT) at 10 h and solid
retention time (SRT) at 80 d, respectively. On this basis, the effects of different SMZ concentrations on
nutrient removal, degradation, and sludge characteristics were compared. The removal efficiency of SMZ
increased with the increase of SMZ concentration. The maximum removal rate was as high as 87%. The SMZ
dosage also had an obvious effect on sludge characteristics. As the SMZ concentration increased, the
extracellular polymer substances (EPS) concentration and the membrane resistance both decreased, which were
beneficial for the reduction of membrane fouling. Finally, seven kinds of SMZ biodegradation intermediates
were identified, and the possible degradation pathways were speculated. The microbial community results
showed that the microbial diversity and richness in the reactor decreased after adding SMZ to the influent. The
relative abundance of Bacteroidetes, Actinobacteria, Saccharibacteria and Nitrospirae increased at the phylum
level. Sphingobacteria and Betaproteobacteria became dominant species at the class level. The relative
abundance of norank-p-Saccharibacteria and Nitrospirae increased significantly, and norank-p-Saccharibacteria
may be the dominant bacteria for SMZ degradation.
Keywords:
IFAS-MBR
Sulfamethazine
Biodegradation
Membrane fouling
Microbial characteristics
The active sludge return system was developed in moving bed biofilm reactor-membrane bioreactor (MBBR-MBR) to form an
integrated fixed-film activated sludge bioreactor (IFAS-MBR) technology [1]. This can not only reduce the content of metabolites, but

also improve the sludge properties to simultaneously slow down and reduce sludge production in membrane fouling. IFAS-MBR also
displayed a higher endurance for shock loading than the MBR [2]. As a novel wastewater treatment process, there has limited report
about antibiotics removal by IFAS-MBR.
As an important component of antibiotics, sulfonamides have been widely detected in urban rivers, effluent from sewage treatment
plants, groundwater and even drinking water [3]. These are most common in municipal sewage treatment plants and pose a serious
threat to water quality and human health [4]. In recent years, the removal methods of trace sulfonamide antibiotics in urban sewage
include activated carbon adsorption [5], resin adsorption [6], ozone catalytic oxidation [7], biological aerated filter [8], constructed
wetland, MBR and so on. But the most effective and economical way is MBR. Some researchers have used MBR to treat
sulfamethazine (SMZ) wastewater to study the long-term effects of different concentrations of SMZ on its degradation efficiency and
sludge characteristics [9]. A lab-scale anaerobic/hypoxia/oxygen membrane bioreactor (A1/A2/O-MBR) was used to study the removal
performance of nine sulfonamides (SAs) at ambient concentrations [10]. However, the complete pathway and degradation mechanism
of SMZ in IFAS-MBR require further study.
Therefore, the objective of this study is to investigate the performance and degradation mechanism of SMZ from wastewater using
IFAS-MBR. Membrane fouling in the reactor with different SMZ concentration was studied by extracellular polymeric substances
(EPS) and soluble microbial products (SMP). Moreover, the intermediate products of SMZ degradation were analyzed by LC-MS and
the microbial community characteristics were determined by high-throughput sequencing.
The schematic structure of the laboratory scale reactor is shown in Fig. S1 (Supporting information). The IFAS-MBR consisted of an
MBBR unit (R1) and an MBR unit (R2). Detailed introduction of reactor and SMZ is in Supporting information (Fig. S2). In this study,
three different hydraulic retention times (HRTs) (6, 8 and 10 h), three different solid retention time (SRTs) (20, 40 and 80 d) and three
different SMZ concentrations (50 μg/L, 100 μg/L, 150 μg/L) were used to compare the performance of IFAS-MBR.
The specific measurement method is shown in the Supporting information. Chemical oxygen demand (COD) and ammonia nitrogen
+
(NH4 -N) were detected using Hach methods 8000 and 10031, respectively [11]. Solid phase extraction was required before HPLC can
be used to determine the SMZ content [12,13]. Quantitative analysis was performed by the external standard method [9]. LC-MS was
used to identify the biological metabolic intermediates of SMZ [14,15]. The total extracellular polymeric substances (T-EPS, include
EPS and SMP) were extracted by the method of cation exchange resin (CER, Sigma-Aldrich, USA) [16]. The contents of the EPS and
SMP were characterized using total organic carbon (TOC), DNA, protein, and polysaccharide. The resistance in series model has been
adapted to consider membrane fouling [17]. The total filtration resistance was defined as the following (Eq. 1):
Rt = Rc + Rf + Rm
(1)
where Rt is the total filtration resistance; Rm is the intrinsic membrane resistance; Rf is the internal fouling resistance due to fouling
mechanisms, and Rc is the cake layer resistance that can be removed by physical cleaning.
Sludges were taken from MBBR and MBR reactors with SMZ influent concentrations of 0, 50, 100, 150 μg/L, numbered
SMZ_0_MBBR, SMZ_0_MBR, SMZ_50_MBBR, SMZ_50_MBR, SMZ_100_MBBR, SMZ_100_MBR, SMZ_150_MBBR,
SMZ_150_MBR, respectively. High-throughput sequencing was performed according to the Illumina platform sequencing process.
The performance of IFAS-MBR at different HRTs is illustrated in Fig. 1a. When SMZ was not added to the influent, the removal
+
efficiencies of COD and NH4 -N were 96.5% and 99.65%, respectively, when the HRT was 8 h. When SMZ was added to the influent,
+
the removal efficiencies of COD and NH4 -N were approximately 89%–92% and 95.5%–97.34%, respectively. It can be observed that
+
the removal rates of COD and NH4 -N were decreased when SMZ was added into the bioreactors. It may relate to the toxicity of SMZ
to the microorganisms that degrade COD. The amino groups in the structure of SMZ degradation were separated and existed in the
+
solution as NH4 , which increased the concentration of ammonia nitrogen. The removal efficiencies of SMZ at different HRT were
approximately 63.5%-73.5%, which increased as HRT increased. This suggested that at the condition of long HRT, the SMZ removal
efficiency was significantly positive. Previous results have shown that the prolongation of HRT reduced the tendency for membrane
pollution and promoted the diversity, symbiosis, and biodegradation efficiency of some micro-pollutants [18]. This study draws the
same conclusion, which may be due to the higher adsorption and biodegradation of SMZ at longer HRT. Since the major removal
mechanism of the system was biodegradation, the attachment growth mode was ideal for enriching slow-growing bacteria and
culturing specific degrading bacteria. Thereby an effective removal of micro-contaminants was obtained.
The performance of IFAS-MBR at different SRTs is shown in Fig. 1b. When SMZ was not added to the influent, the removal
+
efficiencies of COD and NH4 -N were approximately 93.5%–97.2% and 99.1%–99.7%, respectively. The removal efficiencies of COD
+
+
and NH4 -N were very higher at the SRT 80 days. When SMZ was added to the influent, the removal efficiencies of COD and NH4 -N
+
were approximately 89%–93% and 95%–97%, respectively. The removal efficiencies of COD and NH4 -N were reduced, compared
with the SMZ was not added. The possible reasons are the same as discussed above. The removal efficiency of SMZ increased from
69.5% to 78.5% as SRT increased. This also means that longer SRT is more conducive to the survival of slow-growing bacteria and the
adaptation of some SMZ-degraded microorganisms to degrade certain persistent pollutants [19].
The performance of IFAS-MBR at different SMZ influent concentrations is presented in Fig. 1c. The removal efficiencies of COD
+
and NH4 -N were approximately 89%–94% and 95%–99%, respectively. Previous studies suggested that the addition of SMZ had no
+
significant influence on the removal of COD and NH4 -N at high SMZ concentration (1-5 mg/L) [9]. However, in this study, the
+
removal of COD and NH4 -N was reduced. This can due to the simulated domestic wastewater with a low concentration of SMZ at 50-
150 μg/L was used in this research. Therefore, the results were different with high SMZ concentration condition in previous research.
+
This indicated that the SMZ concentration had a certain inhibitory effect on the biodegradation and nitrification of COD and NH4 -N.

In addition, the removal efficiency of SMZ increased with the increased of influent concentration.
a
c100
b100
100
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
COD
ammonia nitrogen
SMZ
COD ammonia nitrogen SMZ
COD ammonia nitrogen SMZ
no SMZ, SRT = 20 d
no SMZ, SRT = 40 d
no SMZ, SRT = 80 d
add SMZ, SRT = 20 d
add SMZ, SRT = 40 d
add SMZ, SRT = 80 d
SMZ = 50 μg/L
SMZ = 150 μg/L
SMZ = 100 μg/L
no SMZ, HRT = 6 h
no SMZ, HRT = 8 h
no SMZ, HRT = 10 h
add SMZ, HRT = 6 h
add SMZ, HRT = 8 h
add SMZ, HRT = 10 h
Fig. 1. The performance of IFAS-MBR under different HRTs (a), different SRTs (b), different SMZ influent concentrations (c). The values are average of 30
sample values.
The characteristics of EPS and SMP are shown in Fig. 2. The concentrations of the four components (TOC, DNA, protein and
polysaccharide) in the EPS and SMP decreased with increasing SMZ concentration. Previous researchers believed that EPS and SMP
were the significant causes of membrane fouling [20]. It has been reported that EPS and SMP have an important impact on the
characteristics and function of the sludge. Among them, EPS has a major impact on the removal of some micro-pollutants. The causes
of membrane fouling may be related to functional bacteria in sludge, which will be discussed below. The membrane resistances results
were shown in Table S1 (Supporting information). The results showed that all the resistance (R, Rf, Rc) also decreased as SMZ
concentration increased. Most of the filtration resistance was attributed to Rf (70%–82%). The proportion of Rf relative to R clearly
increased as the SMZ concentration decreased. This may due to the lower EPS concentration at high SMZ concentration condition. Rc
accounted for 17%–24% of R in the reactors, and the ratio increased with increasing SMZ concentration.
a
b10
10
8
0 μg/L
0 μg/L
50 μg/L
100 μg/L
150 μg/L
50 μg/L
100 μg/L
150 μg/L
8
6
4
2
0
6
4
2
0
de
polysacchari
TOC
DNA
TOC
DNA
proteins
polysaccharide
proteins
Fig. 2. EPS (a) and SMP (b) characterized by DNA, TOC, protein and polysaccharide in the MBR at different SMZ influent concentrations. The values are
average of 30 sample values.
The LC-MS analysis of the main reaction intermediates is shown in Fig. S3 (Supporting information). Analysis of each
corresponding substance revealed that the substance with an m/z of 217 was produced by removing sulfur dioxide from SMZ. The
substance with an m/z of 124 was a product of a bond between sulfate and nitrogen. The hydroxylated product of the pyrimidine ring
had an m/z of 146. The material with an m/z of 295 was derived from the hydroxylation of SMZ. Because these types of intermediate
products were not stable, the measurements of these products were difficult, and research is currently conducted only by inference.
Based on the detected intermediates, the speculated degradation pathway of SMZ in the IFAS-MBR reactor is shown in Fig. 3.
Fig. 3. Degradation pathway of sulfamethazine in the IFAS-MBR.
The microbial community results are shown in Table S2 (Supporting information). The diversity and richness of the microorganisms
decreased after SMZ was added in the reactor, and the addition of SMZ inhibited some microorganisms. As shown in Fig. 4a,
Proteobacteria and Bacteroidetes were the dominant phyla in the reactors with different SMZ influent concentration, which were
similar to other microbial communities, such as soil and sewage [21]. Proteobacteria decreased from 63.27% to 41.27% while

Bacteroidetes increased from 9.50% to 43.51%, when 50 μg/L SMZ was added in the influent. It has been reported that Proteobacteria
included a variety of functionally important bacteria for organic and nitrogen removal [22]. Thus, the higher abundance of
Proteobacteria could account for the slightly better removal of pollutants in the SMZ-0 than in the SMZ-50. It is reported that
Bacteroidetes could potentially release more membrane foulants such as proteinaceous EPS [23]. Also, Bacteroidetes possess fimbriae,
which could help them attach to the supporting material surfaces [24]. After the addition of SMZ, faster membrane fouling occurred in
the SMZ-50 due to the relatively high abundance of Bacteroidetes in the sludge. However, the content of Bacteroides in SMZ-100 and
SMZ-150 did not change much, which also indicated that the membrane fouling not only be affected by Bacteroides.
After the addition of SMZ, Sphingobacteria and Betaproteobacteria became the most abundant dominant species (Fig. 4b). It has
been reported that these specific bacteria were the pioneers of surface colonization on membranes [25]. It can be stated that these
shared dominant bacteria may be responsible for the initial membrane fouling. The relative abundance of Saccharibacteria in SMZ-150
was significantly increased at the level of the phylum, while the relative abundance of norank-p-Saccharibacteria also increased
significantly at the level of the class. The norank-p-Saccharibacteria has been reported to play an important role in promoting the
biosynthesis of antibiotics, the biosynthesis of secondary metabolites and the degradation of pyrimidines [26]. The norank-p-
Saccharibacteria may be the dominant bacteria for SMZ degradation.
a SMZ-150-MBR
bSMZ-150-MBR
SMZ-150-MBBR
SMZ-100-MBR
SMZ-100-MBBR
SMZ-50-MBR
SMZ-50-MBBR
SMZ-0-MBR
SMZ-150-MBBR
SMZ-100-MBR
SMZ-100-MBBR
SMZ-50-MBR
SMZ-50-MBBR
SMZ-0-MBR
SMZ-0-MBBR
SMZ-0-MBBR
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
Percent of community abundance on phylum level
Percent of community abundance on class level
Proteobacteria
Acidobacteria
Planctomycetes
Chlamydiae
Bacteroidetes
Gemmatimonadetes
Nitrospirae
Chloroflexi
Actinobacteria
Gammaproteobacteria
Alphaproteobacteria
Thermomicrobia
Norank-p-Saccharibacteria
KD4-96 Planctomycetacia
Sphingobacteria
Actinobacteria
Gemmatimonadetes
Betaproteobacteria
Saccharibacteria
Firmicutes
Others
Flavobacteriia
Acidobacteria
Verrucomicrobia
Deltaproteobacteria
Latescibacteria
Cytophagia
Nitrospira
OM190
Caldilineae
Others
Anaerolineae
Fig. 4. Analysis of microbial diversity at a different taxonomic level for different SMZ influent concentrations samples: phylum (a); class (b).
In summary, when treating synthetic wastewater in the IFAS-MBR reactor at different HRT and SRT operating conditions, the
+
removal rates of COD and NH4 -N decreased after the addition of SMZ. For the SMZ removal effect, the optimal HRT and SRT were
10 h and 80 d, respectively. The biodegradation rate of SMZ increased as the influent SMZ concentration increased. The concentration
of EPS and SMP decreased with the increase of SMZ concentration. The results showed that increasing the SMZ concentration can
slow down membrane fouling. Seven kinds of SMZ biodegradation intermediates were identified by the analysis, and the possible
degradation pathways were speculated. After the addition of SMZ, the relative abundance of Bacteroidetes, Actinobacteria,
Saccharibacteria and Nitrospirae increased significantly at the level of the phylum. Faster membrane fouling occurred in the SMZ-50
due to the relatively high abundance of Bacteroidetes in the sludge. Sphingobacteria and Betaproteobacteria became the most abundant
dominant species. The norank-p-Saccharibacteria may be the dominant bacteria for SMZ degradation.
Acknowledgments
This work was financially supported by the Major Science and Technology Program for Water Pollution Control and Treatment
(Grant No.2018ZX07601-003) and the Fundamental Research Funds for Central Public Research Institutes of China (Grant No. 2019-
YSKY-009).
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