Comparison of novel functionalized nanofiber forward osmosis membranes for application in antibacterial activity and TRGs rejection

Journal Pre-proof Comparison of Novel Functionalized Nanofiber Forward Osmosis Membranes for Application in Antibacterial Activity and TRGs Rejection Haisheng Chen (Data curation) (Formal analysis) (Investigation) (Writing - original draft) (Methodology), Shengyang Zheng (Formal analysis) (Methodology), Lijun Meng (Formal analysis) (Methodology), Gang Chen (Writing - review and editing), Xubiao Luo (Writing - review and editing), Manhong Huang (Conceptualization) (Formal analysis) (Funding acquisition) (Resources) (Supervision) (Validation) (Writing - review and editing)

PII:

S0304-3894(20)30238-7

DOI:

https://doi.org/10.1016/j.jhazmat.2020.122250

Reference:

HAZMAT 122250

To appear in:

Journal of Hazardous Materials

Received Date:

12 November 2019

Revised Date:

3 February 2020

Accepted Date:

4 February 2020

Please cite this article as: Chen H, Zheng S, Meng L, Chen G, Luo X, Huang M, Comparison of Novel Functionalized Nanofiber Forward Osmosis Membranes for Application in Antibacterial Activity and TRGs Rejection, Journal of Hazardous Materials (2020), doi: https://doi.org/10.1016/j.jhazmat.2020.122250

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Comparison of Novel Functionalized Nanofiber Forward Osmosis Membranes for Application in Antibacterial Activity and TRGs Rejection

Haisheng Chen

a,d

, Shengyang Zheng a, Lijun Meng a, Gang Chen a, Xubiao Luo

c,, ,

Manhong Huang a,b,,

a

College of Environmental Science and Engineering, State Environmental Protection

Engineering Center for Pollution Treatment and Control in Textile Industry, Donghua

b c

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University, Shanghai, 201620, China

Shanghai Institute of Pollution Control and Ecological Security, Shanghai, 200092, China Key Laboratory of Jiangxi Province for Persistent Pollutants Control and Resources

Aerospace Kaitian Environmental Technology Co., Ltd, Changsha, 410100, China

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d

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Recycle, Nanchang Hangkong University, Nanchang, 330063, China



Corresponding author: Manhong Huang, College of Environmental Science and

Engineering, Donghua University, Shanghai, 201620, People’s Republic of China; Phone: +86 21 67792546; Fax: +86 21 67792546; E-mail: [email protected]

Graphical Abstract 1

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Highlights

The AgNPs was anchored on TiO2 to form antibacterial TiO2/AgNPs nanoparticles.



FO membranes functionalized with nanoparticles were fabricated by eletrospinning.



TFN2 membrane exhibited more excellent water flux and antibacterial properties.



Most E. coli cells in contact with the TFN2 membrane began to rupture and die.



TRGs on the transposons and integrons are easier to penetrate the membrane.

Abstract

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Antibiotic resistance genes (ARGs) are serious pollutants in municipal sewage treatment plants and may cause significant harm to ecological systems, microbial fouling is also inevitable in membrane process. Herein, novel forward osmosis (FO) membranes made of electrospun nanofibers (TFN0) and further impregnated with titanium dioxide (TiO2) (TFN1) nanoparticles and titanium dioxide/silver composite nanoparticles (TiO2/AgNPs) (TFN2). The FO membranes were used to compare the antimicrobial performance and rejection of tetracycline-resistant genes (TRGs). Characterizations revealed that the TiO2/AgNPs were 2

evenly scattered in the polysulfone (PSf) nanofibers and resulted in a TFN2 membrane that exhibited excellent physicochemical properties, filtration, and antibiofouling performance in real wastewater. The cell viability analysis revealed that the antibacterial effect of the TFN2 membranes was significantly better than that of TFN1, as indicated by about 65% of E. coli cells killed after contact with the TFN2 membrane. TFN2 membranes had greater rejection rates of TRB and TRGs than TFN1. The TRG permeation rates of the TFN2 membrane in the FO mode (active layer facing the feed solution) were 39.62% and 33.02% lower than the TFN0 and TFN1 membranes, respectively. FO membranes modified by the TiO2/AgNPs

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nanocomposites hold promise to remove ARGs and pathogens from wastewater treatment plant effluents.

Keywords: Electrospun nanofiber-supported FO membrane; TiO2/AgNPs; antimicrobial

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activity; tetracycline resistance; antibiofouling 1. Introduction

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As typical micro-pollutants, antibiotic resistance genes (ARGs) pose potential threats to human health and ecosystems [1, 2]. To date, ARGs have been widely found in surface water,

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groundwater, and medical and livestock wastewater [3, 4]. Wastewater treatment plants (WWTPs) contain a large number of ARGs because of the introduction of antibiotics from

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sources such as hospitals, agriculture, and industry [5]. Such treatment plants are considered to be a main genetic reactor for ARGs [6, 7]. For instance, tetracycline, an important antibiotic, causes a leakage of tetracycline-resistant bacteria (TRB) and even tetracycline-

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resistant genes (TRGs) [8]. In addition, activated sludge processes are widely used in WWTPs, leading to an accumulation of antibiotic resistance bacteria (ARB), ARGs, and

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related mobile elements [9]. WWTPs are the main source of ARB and ARGs release into the environment, and they are considered hotspots for broader ARG dissemination [10]. In recent years, advanced oxidation processes such as ozonation, chlorination, and UV disinfection have been used to reduce TRGs in WWTP wastewater [11]. Although these methods can effectively inactivate ARB, they may also increase the abundance of extracellular ARGs in wastewater [12]. 3

Physical treatment methods such as membrane treatment have increasingly been used for ARGs removal [13, 14]. Li et al. [15] developed an integrated process of pre-coagulation and microfiltration that effectively reduced the absolute abundances of ARGs (>2.9 logs) from effluent. As a popular research topic of membrane treatment technology in the field of sewage treatment, forward osmosis (FO) technology is very suitable for application in the removal of ARGs and other micropollutants. Although FO cannot usually be applied to large scale wastewater treatment, FO membrane technology offers great advantages in the water reuse field [16, 17], and it can be used to treat effluent from the secondary clarifier of

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WWTPs to achieve retention of ARGs for water reuse. FO does not require external pressure as the driving force, and it produces little or no secondary pollution. FO tends to obtain clean water from a variety of complex water environments [18].

To achieve high permeation flux, high rejection, and antifouling performance, more

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attention has been paid to the polyamide FO membranes with thin film composite (TFC) structure [19, 20]. However, TFC membranes suffer from biofouling during FO processes

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[21] that reduce the filtration performances and solute rejection. To improve the antibacterial property, embedding or impregnating antimicrobial nanomaterials into the polymeric matrix

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of membranes is one of the most effective solutions. Pan et al. [22] embedded Ag nanoparticles (AgNPs) into the electrospun PAN nanofiber TFC FO membrane, which

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exhibited excellent bacterial inactivation rates (>90%) for two common bacteria. In addition to AgNPs, titanium dioxide (TiO2) nanoparticles are also commonly used antibacterial materials [23]. Due to the large band gap (3.2-3.5 eV), TiO2 only absorbs UV irradiation, and

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the electron hole recombination limits its photocatalysis and antibacterial activity [24]. Thus, chemical modifications such as element doping or conjugation are used to increase the light

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absorbance and photocatalytic activity [25]. For example, Jatoi et al. [26] conjugated AgNPs with TiO2 nanoparticles and subsequently blended the catalyst into the cellulose acetate (CA) nanofiber matrix, which was then shown to inhibit bacterial growth. AgNPs can play a role of electron receptor, suppressing the recombination of hole pairs and photogenerated electrons, and thus realizing the antibacterial properties of TiO2 in the absence of ultraviolet radiation [27, 28]. Moreover, AgNPs compromise bacterial cell integrity and metabolism through released dissolved silver species or the production of reactive oxygen species (ROS) 4

[29]. Therefore, Ag-doped TiO2 NPs (TiO2/AgNPs) could potentially be used to improve the antimicrobial property of FO membranes and ARG removal. However, the previous studies have only focused on the study of simulated antibiotic wastewater with

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TiO2/AgNPs-modified FO membrane [30] and did not discuss the antibacterial performance of different particle membranes; thus actual wastewater treatment was not involved. Therefore, we further explore this area. In this work, polysulfone (PSf) with stable physical and chemical properties was selected as the spinning polymer, and PSf, PSf/TiO2 and PSf/TiO2/AgNPs nanofiber

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membranes were prepared via an electrospinning technique, which then formed a polyamide TFC FO membrane. To explore the morphology and structure of the prepared samples, X-ray diffraction

(XRD),

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field

emission

scanning

electron

microscope

(FESEM),

Fourier-transform infrared spectroscopy (FTIR), and high-resolution transmittance electron

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microscopy (HRTEM) were used. Permeate flux experiments and bacterial viability tests were carried out to evaluate the pollutant rejection and antibiofouling behavior of different

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FO membranes. Finally, the FO membrane performance on the interception of tetracycline resistance (TRB and TRGs) collected from real wastewater effluent was investigated to shed

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new light onto the development of next-generation membrane filtration systems toward antimicrobial fouling and TRGs.

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2.1. Materials

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2. Materials and Methods

Polysulfone (PSf, molecular weight∼22000), TiO2 (particle size<50nm, 99.7%), N, (DMF,

98%),

1-methyl-2-pyrrolidinone

(NMP,

98%),

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N-Dimethylformamide

M-phenylenediamine (MPD, >99%), and 1, 3, 5-trimesoyl chloride (TMC, 98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dopamine hydrochloride (Dopa, 98%) and tris HCl buffer (1M, PH=8.5) was purchased from J&K Scientific. Isopar-G (99.9%) as a solvent was received from Exxon Mobil. The Sinopharm Group, China supplied the silver nitrate (AgNO3: 99.8%) and sodium chloride (NaCl, >99.5%). Escherichia coli (E. coli) bacterial slope was obtained from the Shanghai Luwei Technology Co., Ltd. 5

The wastewater containing TRB was the effluent of secondary clarifier of a WWTP in Shanghai, China. Water quality of the wastewater is shown in Table S1. SYBR® Green PCR Master Mix, double distilled water, and primers for real-time quantitative PCR detecting System (QPCR) were purchased from Sangon Biotech, Shanghai, China. The target genes and its primer information for QPCR are shown in Table S2. 2.2. Preparation of TiO2/AgNPs nanocomposite particles and membranes To synthesize TiO2/AgNPs nanocomposite particles, dopamine or Dopa was used to

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reduce Ag+ to AgNPs. Polydopamine (pDopa) was formed by polymerization to bind AgNPs and TiO2 together. The chemical structure of Dopa and pDopa are shown in Fig. S1. Briefly, Dopa was added to tris HCl buffer to form a 2-mg/mL Dopa solution, followed by proper doses of TiO2 nanoparticles and rigorous mixing for 18 h. After high-speed centrifugation,

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TiO2 nanoparticles with pDopa were produced and then added to 350 mL of AgNO3 solution (0.2 M). The suspension was stirred vigorously overnight and centrifuged again, and finally

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the precipitation of TiO2/AgNPs nanoparticles was prepared.

The PSf nanofiber membrane and its modification were operated in the following

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procedures. In a mixed solution of 7 mL DMF and 3 mL NMP, 0.3 wt.% TiO2 and TiO2/AgNPs nanoparticles were dispersed and spread evenly by ultrasonicator. After adding

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26 wt.% PSf to this solution, it was stirred at 1000 rpm for 8 h. This solution was employed as the spinning solution and loaded onto the spinning device. The electrospinning parameters are listed in Table S3. The nanofiber membrane substrates were prepared by 4-mL spinning

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solution and can be used for interfacial polymerization after drying. The substrates were named PSf0, PSf1, and PSf2, respectively, corresponding to a blank sample blended with

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TiO2 and TiO2/AgNPs nanoparticles. The dense film of polyamide (PA) layer was formed by the reaction of the MPD and

TMC solutions. The nanofiber membrane substrates were immersed in 2.0-wt.% MPD solution for 120s. The isopar-G solution containing 0.15 wt.% TMC was then carefully poured onto the membrane surface for 60 s to fully react with the MPD to form the PA layer. The FO membrane was dried at 85 °C and stored in 4 °C pure water before use. The FO 6

membranes prepared from PSf0, PSf1, and PSf2 were named TFN0, TFN1 and TFN2, respectively. 2.3. Characterization of nanoparticles and membranes The morphology and composition of the samples were characterized by FESEM (Hitachi S-4800, Japan), TEM (JEM-2100, Japan), HRTEM (*/Talos F200S, Japan), and XRD (D/max-2550VB+/PC, Japan). The main functional groups of the active layer on the FO membranes were measured on a FTIR (Nicolet 6700, USA). The surface hydrophilicity

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change caused by the hydrophilicity effect of the nanoparticle addition in the different PSf nanofiber membranes and the FO membranes were determined by a contact angle goniometer (Model SL200C, China) [31]. The average nanofiber diameter was determined

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by a statistical analysis of the SEM images with image processing software Image J. 2.4. Forward osmosis system

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As displayed in Fig. S2, the permeability of the FO membranes was measured by laboratory-made FO equipment. A membrane module with an actual membrane area of 12

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cm2 was driven by gear pumps (WT3000-1FA, Longer, China) to drive the feed solution and draw solution. 1M NaCl solution was used as the draw solution, the secondary effluent from

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the Songjiang sewage treatment plant was taken as the feed solution, and the volume was 1 L. Before the FO experiment began, the draw solution, membrane module, plastic tube, and beakers were sterilized in an autoclave at 120 °C for 30 mi, and experimental temperatures

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were kept at 25.0 ± 0.1 °C. To obtain the water flux (Jw, L·m−2·h−1, abbreviated as LMH) of the FO membranes, the computer automatically recorded the changes in the quality change

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of draw solution every 5 min, and the conductivity of the feed solution was monitored at all times.

After the experiment, an appropriate amount of draw solution was taken to measure the

water quality (TP, TN, NH4+-N, COD, TOC and SS) and compared with the secondary effluent. Through this comparison, the interception rate of each water quality index was calculated to determine the purification effect of the FO membranes on water quality. 7

High-throughput sequencing technology was used to analyze the microbial community structure in the draw solution. Jw was evaluated from the following equations: J𝑤 =

∆𝑚 1000𝐴∆𝑡

,

(1)

where A (m2) is the actual area of water through the membrane and m (g) is the increased weight of draw solution at a certain time t (h); 2.5. Membrane antimicrobial activity

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2.5.1 Diffusion inhibition zone test An antibacterial circle experiment of various FO membranes was conducted as follows. 100 μL of the E. coli bacterial suspension (108 CFUmL-1) was spread on beef agar medium. After UV sterilization, the circular FO membranes were placed on the agar plate. The solid

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medium was placed in a 37 °C incubator for 24 h to allow the bacteria to proliferate. The proliferation of bacteria around the membrane in agar plates was photographed with a digital

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camera. To obtain the diffusion inhibition zones, software Image J was used to measure the

2.5.2 Cell viability analysis

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size of the bacterial growth inhibition zone.

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Antibacterial activity of the TFN0, TFN1, and TFN2 membranes were compared by analyzing the number of viable cells that survived on the membrane. The FO membranes (4 cm×4 cm) were spread flat in an empty petri dish, and 200 μL of the bacterial inoculums

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(108 CFUmL-1) were added to infiltrate the membrane evenly. After 2 h under appropriate

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conditions, the membrane samples was removed and 10 mL of saline was added. The bacteria were separated from the membrane after 15 min of ultrasonic treatment. The bacterial fluid was diluted to three concentration gradients, and the number of bacteria was counted after being cultured in the beef agar medium at 37 °C overnight by plate coating method. Three parallel samples were thus prepared for all tests. In addition, changes in the bacteria cell structure on the FO membranes surface were examined. The membrane samples were treated with 5% glutaraldehyde and a gradient of 8

ethanol before acquiring the SEM images. 2.6. Rejection of TRB and TRGs The interception effect of the TFN0, TFN1, and TFN2 membranes on tetracycline resistance was represented by the TRB and TRGs permeability. In the FO mode (PA layer facing the feed solution) and the PRO mode (PA layer facing the draw solution), three kinds of FO membranes were used for FO experiment. Concentrations of TRB and TRGs in the secondary effluent and the draw solution after the 2 h experiment were detected. A water

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sample of 5 mL was added to 45 mL of phosphate buffered saline and shaken for 2 h (180 rpm). The amount of TRB was determined by coating on beef agar solid medium containing 16 mg/L of tetracycline [32].

The DNA of water samples was stored in Tris-EDTA buffer solution and kept at -20 °C.

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Seven d genes were quantitatively detected by the SYBR® Green method [33]. A reaction solution containing 20-μL PCR Master Mix, primer and DNA sample was executed in

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LightCycler480 Software Setup (Roche). The copy number of genes was calculated by standard curve. In order to reduce the error, three sets of parallel samples were set for each

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DNA sample, and R2 values were more than 0.99, respectively. A one-way analysis of variance (ANOVA) acquired by IBM SPSS Statistics 20.0 software was used to characterize

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the relationship between membrane types and the permeation rates of seven genes in two modes. The permeation rates of TRB (P1, %) and TRGs (P2, %) were evaluated from the

𝐵𝑉 𝐵0 𝑉0

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𝑃1 =

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following equations:

𝑃2 =

(2) 𝐶𝑉

,

𝐶0 𝑉0

(3)

where B (CFUmL-1) and C (copiesmL-1) are TRB and genes concentrations in the draw solution after 2h; B0 (copiesmL-1) and C0 (CFUmL-1) are initial TRB and genes concentrations in the secondary effluent; V (L) represents the volume of NaCl solution after 2h; and V0 (L) represents the volume of the secondary effluent before the experiment. 3. Results and Discussion 9

3.1. Material characterization 3.1.1 Nanoparticles characterization Figure 1a shows the specific form of the TiO2 nanoparticles in TEM. TiO2 nanoparticles are spherical and rod-like shaped with sizes between 20 nm ~ 45 nm. Compared to the TiO2 nanoparticles, the TiO2/AgNPs nanocomposite particles are surrounded by pDopa as shown in Fig. 1b. It can be clearly seen that the TiO2 nanoparticles are coated with clustered pDopa, and their surface is also covered with AgNPs. The HRTEM image of the AgNPs in Fig. 1c

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reveal AgNPs of 2 nm ~ 6 nm, much smaller than the TiO2. Furthermore, (111) lattices of the AgNPs are identified by the 0.241 nm of the lattice distance, indicating that Dopa had successfully reduced Ag+ to AgNPs as reported previously [34].

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3.1.2 Characterization of membranes

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TEM micrographs were used to observe the distribution of nanoparticles in nanofibers. Fig. 2 shows that TiO2 nanoparticles (Fig. 2b) and TiO2/AgNPs (Fig. 2c) nanocomposite

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particles were embedded in the PSf nanofiber (Fig. 2a). The TiO2 nanoparticles in nanofibers appeared to agglomerate, whereas the TiO2/AgNPs are dispersed well due to the pDopa coating. The element mapping indicates that the nanoparticles are uniformly distributed in

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nanofibers (Fig. 2d and 2e), which confirms that the nanoparticles are fully loaded in PSf nanofibers. In addition, Fig. 2e also demonstrates the existence of a large number of AgNPs,

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consistent with the abovementioned conclusions. Figure 3 exhibits the SEM images and fiber diameter distribution of the PSf0, PSf1 and

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PSf2 nanofiber substrates. Uniform and smooth nanofibers can be observed on all nanofiber substrates. In addition, the fiber membrane is highly porous with interconnected pores, leading to a lower internal concentration polarization (ICP) and greater mass transfer rates [35]. As shown by the fiber diameter diagram, the diameter of nanofibers increased gradually with the addition of nanoparticles. The nanofiber substrate has the largest fiber diameter, reaching 979.79 ± 119.31 nm. Electrospinning technology for preparing the nanofibers from polymer solutions required the application of sufficiently high electrostatic 10

pressure fields to overcome the tension of the droplets [36]. When the nanoparticles were blended with PSf, the viscosity of the spinning solution was higher, thereby increasing the fiber diameter [37]. Moreover, nanoparticles are generally embedded in the interior of the nanofiber, which also increased the nanofiber diameter as verified in TEM micrographs in Fig. 2. In order to study the phase and crystalline nature of the composite nanofibers, an XRD analysis of the composite nanofiber substrates was carried out (Fig. 4). A few broad noncrystalline peaks at about 15–25° is observed in all samples, which correspond to the

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characteristic peaks of pure PSf. In the PSf1 nanofiber substrate, diffraction peaks at the 2θ values of 25.32°, 37.86°, 48.06°, 53.97°, 55.09° and 62.75° are observed, and these peaks represented the (101), (004), (200), (105), (211), and (204) anatase TiO2crystal planes,

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respectively, according to the International Center for Diffraction Data (ICDD) card (card no.4-783). The same peaks are also observed on the PSf2 nanofiber substrate. In addition,

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the characteristic peaks at 2θ of 38.12°, 44.28°, 64.43° and 77.47° are observed, which correspond to the (111), (200), (220), and (311) cubic crystal AgNPs [38]. The results of

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XRD also confirm the impregnation of TiO2/Ag nanoparticles into the nanofiber material. The FTIR spectrum indicates that interfacial polymerization reaction occurred. As shown in Fig. 5a, there is an extremely compact and rough PA layer, as interfacial

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polymerization typically creates a typical ridge-valley structure [39, 40]. According to the sectional images (Fig. 5b and c), the thickness of TFN2 membrane is about 70 μm, including

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a PA layer, a PSf nanofiber substrate, and a polyethylene terephthalate support. Such a multilayer porous composite structure facilitates the mass transfer of water and salt [41]. The

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FTIR spectroscopy results in Fig. 5d revealing significant peaks at the wave numbers of 1146cm−1 (S=O symmetric stretching), 1272 cm−1 (Aryl-O-aryl C–O stretch), and 1410 cm−1 (C=C aromatic ring stretching), which match the specific functional groups of PSf [42]. The apparent absorption peaks of the polyamide layer are presented at 1540 cm-1 (−N−H, amide II peak), 1610 cm-1 (aromatic ring breathing of amide), and 1664 cm-1 (−C=O, amide I peak), as observed on each FO membrane [43]. Surface hydrophilicity is critical to improving water flux and contamination resistance 11

during membrane separation. Since the PSf is a hydrophobic material, the water contact angle of a pure PSf substrate is 130.33±0.84°. Figure 6 shows that after two kinds of nanoparticles were added respectively, the hydrophilicity of the substrates is enhanced and the water contact angle is reduced, mainly because of strong hydrophilicity of nanoparticle and the relatively hydrophobic aromatic rings in pDopa [44]. TiO2/AgNPs nanocomposite particles increase the hydrophilicity of nanofibers more than the TiO2 nanoparticle. A similar phenomenon also occurred in the hydrophilicity changes of the FO membranes. Due to the successful formation of PA active layer on nanofiber membrane, the

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contact angle of the FO membranes rapidly decreased. A considerable decrease in contact angle (from 77.60±1.10° to 63.06±0.70°) was noticed in the TFN2 membrane. Moreover, the membrane surface roughness also increased, which may have facilitated the

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membrane-water interactions and thus reduced the water contact angle [45]. The TiO2/AgNPs were rich in hydrophilic groups, so the water flux and antibiofouling

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performance of the FO membrane was greatly improved [46, 47].

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3.2 Antibacterial activity of FO membranes 3.2.1 Diffusion inhibition zone test

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The results of the antibacterial circle experiment of various FO membranes in Fig. 7 indicates that the TFN2 membrane exhibits a remarkable inhibition effect as characterized by

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the 0.99±0.03 mm hollow ring without bacterial growth. Conversely, the pristine FO membrane (TFN0) does not result in any inhibition. Moreover, the TFN1 membrane also

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exhibits negligible antibacterial activity, probably because TiO2 causes some antibacterial properties only under UV irradiation [48], whereas our experiments were carried out under room light or visible light. Our results confirm that the TiO2/AgNPs modified membrane strongly inhibits the growth of E. coil bacteria, which is attributed to ROS and Ag+ released into the LB medium from the TFN2 membrane. 3.2.2 Cell viability analysis 12

The phenomenon in Fig. 8 illustrates that the bacterial cell structure exhibits significantly different changes after contact with the FO membrane. The cells attached to the TFN0 and TFN1 membranes have a relatively intact structure and a smooth surface (Fig. 8a and b), while bacteria on the TFN2 membrane surface underwent obvious structural damages (e.g., cracks and pits as shown in Fig. 8c), owing to the presence of the TiO2/AgNPs particles. After exploring the survival of E. coli cells on the FO membranes for 2 h, the antimicrobial efficiency of the three membranes is compared in Fig. 8d. Different from the TFN0 membrane, the TFN2 membrane results in a death ratio of 65% of the E. coli cells. In

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contrast, TiO2 nanoparticles on the TFN1 membrane killed only less than 20% of the E. coli cells.

3.2.3 Antimicrobial Mechanism of TiO2/AgNPs modified FO membrane

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The process by which the TiO2/AgNPs modified membrane produce an antibacterial

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effect is displayed in Fig. 9. TiO2 produce ROS under ultraviolet irradiation such as ･O2(superoxide radical), ･OH (hydroxyl radical), ･HOO (peroxide radical), and 1O2 (singlet

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oxygen) that damage cell walls and interfere with membrane functions [49]. However, due to a wide band-gap, TiO2 exhibits low antibacterial activity under visible light on the TFN1 membrane. The addition of silver could shrink the TiO2 band-gap and decrease the

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recombination rate of the electrons and holes [50]. Under the synergy of TiO2 and silver, TiO2/AgNPs will generate a large number of negatively charged electrons (e-) and positively

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charged hole pairs (h+) when excited, e- and h+ react with oxygen (O2) and water (H2O) to form ROS, such as ･O2-, ･OH, ･HOO, and so forth [51]. The following equations (Eqs.

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(4)–(9)) outline the main processes of the reaction. The ROS can directly oxidize organic matter to inorganic molecules, and ･O2- can also attack polyunsaturated phospholipids in the cell structure, so they can destroy bacterial cell walls and block their respiratory systems [52], as shown: TiO2/AgNPs + hv → TiO2/AgNPs (e- + h+) h+ + H2O → ･OH + H+ 13

(4) (5)

O2 + e- → ･O2-

(6)

･O2- + H2O → ･HOO + OH-

(7)

･HOO + H2O → H2O2 + ･OH

(8)

H2O2 → ･OH

(9)

.

In addition, the released Ag+ from the AgNPs in TFN2 membrane may interact with the thiol groups (-SH) of cell wall, cell membrane, and DNA [53] and further destroy the integrity of the bacteria to cause irreversible cell damage. Moreover, Ag+ affected ribosome

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and enzyme function, disrupting DNA to kill cells [54]. More specifically, the TFN2 membrane causes bacteria death by multiple methods including damage structure, ATP synthesis prohibition, deactivation of enzymes, interfering with DNA replication, as well as a synergistic effect of TiO2 and Ag nanoparticles. In addition, the conjugation of AgNPs

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with TiO2 may prevent excessive release of Ag+ and lengthen the antibacterial performances [55]. To monitor the leaching of silver, the silver concentration detected in the solution after

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the FO experiment was lower than the detection limit (0.1ppb), indicating little leaching of silver, which also proved that fixing AgNPs on TiO2 and embedding them in nanofibers can

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effectively reduce the leaching. A similar phenomenon has appeared in some previous studies—that is, blending nanoparticles with nanofibers can effectively reduce particles

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leaching and increase membrane antibacterial life [22, 56].

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3.2.4 Membrane antifouling performance To study the antifouling performance of the FO membranes when treating the actual

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wastewater, the normalized water flux was monitored and is shown in Fig. 10. Figure 10a presents the flux changes during alternative filtration of pure water and secondary effluent. At 120 min, after continuous filtration experiments of pure water and secondary effluent for 60 min each, the fluxes of the TFN0, TFN1 and TFN2 membranes decreased to 0.52, 0.61, and 0.68 of the initial water flux, respectively. In addition, the flux recovery of the TFN2 membrane was the most obvious after 60 min of another pure water filtration experiment, and the flux loss of the TFN2 was the least at 240 min. All of these showed that TFN2 has 14

the best antifouling performance, followed by TFN1 and TFN0 the worst. In Fig. 10b, over the course of filtration, a decline in the water flux was observed for all FO membranes due to the adhesion and deposition of bacteria (as known as biofouling). For example, after 8 h, the water flux on the TFN0 membrane dropped by about 50%. In contrast, the TFN2 membrane had only a 21% decline, which is indicative of greater biofouling resistance. The antibiofouling effect may arise from the presence of TiO2/AgNPs nanoparticles in the TFN2 membrane, which prevented bacteria from adhering to the membrane surface and clogging the membrane pores. Moreover, the antifouling feature may also be due to the enhanced

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surface hydrophilicity and electrostatic interactions on the TiO2/AgNPs-modified FO membrane [57, 58].

3.3.1 Water flux and permeation rates of TRB

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3.3 Tetracycline-resistance removal

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Figure 11 reveals the water flux and permeability of the TRB-containing water on different FO membranes. The TFN0, TFN1 and TFN2 membranes operating in FO mode

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yielded lower water flux than those in PRO mode, because the concentrative internal concentration polarization (ICP) in PRO mode has a higher effective osmotic pressure [59].

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Compared with the TFN0 membrane, the water fluxes of the TFN1 and TFN2 membrane increased in both modes. The TFN2 membrane reached a maximum water flux of 56.49± 3.89 LMH in the PRO mode because of the increase of hydrophilicity and antifouling

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properties derived from the impregnated nanoparticles. When filtering TRB-containing water, the TFN2 membrane exhibited the lowest permeability in two modes. In addition to the FO

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membrane capable of retaining most of the TRB, under the influence of antibacterial TiO2/AgNPs the permeability of the TRB of TFN2 membrane was as low as 0.75±0.14% in FO mode. Compared with the TFN0 membrane, the permeability of the TFN1 membrane to the TRB decreased slightly due to its hydrophilicity and electrostatic repulsion against bacteria [60]. Furthermore, the retention rates of the water quality indexes of different FO membranes 15

were determined using real wastewater. Results in Fig. S3 show that the TFN2 membrane has the best retention effect on TP, TN, NH4+-N, COD, TOC, and SS, which can be attributed to the retention of more bacteria. Bacteria were rich in organic matter, N, P and other substances, and lower bacterial permeability means higher water purification. 3.3.2 Bacteria diversity changes Figure 12 shows the bacterial diversity of the secondary effluent and the three FO membranes. Proteobacteria, Bacteroidetes, and Planctomycetes were the main classes of

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microbial community in the secondary effluent from the sewage treatment plant, which agrees with the literature [61, 62]. Microbial diversity apparently decreased after filtration with different FO membranes in all operating conditions. Proteobacteria was still the dominant class in the draw solution. Notably, after treatment with the TFN1 and TFN2

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membrane, the Euryarchaeota gradually increased in abundance, which may be attributed to differences in the structure and function of the archaeal cells. Many archaeal cell walls

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contain a variety of complex multimers and secrete extracellular polysaccharides to protect against bactericidal substances [63, 64]. Under the same conditions, the Euryarchaeota was

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3.3.3 Permeation of TRGs

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less susceptible to ROS and Ag+, which led to a higher abundance in the draw solution.

To compare the effects of trapping TRGs by the TFN0, TFN1 and TFN2 membranes,

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TRGs, int1 and 16s rRNA were detected by QPCR to obtain their abundance. As shown in Fig. 13a, the permeation rates of all genes are less than 7% in all operating conditions of

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membrane filtration. Clearly, the FO membrane can effectively retain most of the TRGs. TRGs may exist in smaller viruses or bacteria, or they may exist outside the cell in the form of free DNA, which can enter the draw solution through the membrane pores [65]. Furthermore, DNA is a long-chain macromolecule that is more difficult to pass through membranes than water. Based on this, a many studies have shown that membrane processes such as RO, UF, and MBR can effectively intercept most ARGs [66]. In addition, membrane foulants will adhere to the membrane, such as bacterial, insoluble matters, and extracellular 16

polymeric substances [67]. Studies have shown that proper membrane fouling can reduce the effective pore size of the membrane, and the membrane foulants with high molecular weight and cross-linked structural characteristics combine with microbial cells, which is conducive to the retention of more microorganisms or even smaller DNA fragments [68, 69]. In Fig. 14a, the permeation rates of TRGs in FO mode are lower than those in PRO mode. The permeation rates of the TFN2 membrane in two modes were 39.62% and 29.30% lower than those of TFN0, respectively. The relationship between membrane types and the permeation rates of 7 genes in the two modes is listed in Table S4 and confirms a significant difference

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between membrane types and the permeation rates of genes (p=0.01<0.05). Additionally, it is clear that tetA, tetC, tetO, and int1 had much higher permeation rates than others regardless of the operating condition. This implies a potential dependence of TRG rejection on their content and distribution. According to their different functions, TRGs are often classified

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into ribosomal protection genes, efflux pump genes, and enzymatic inactivation genes [70]. Previous research has specifically defined that the efflux pump genes (tetA, tetC, and so

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forth.) have the highest abundance in general sewage containing TRGs [71, 72]. Additionally, the vast majority of TRGs are located on plasmids [73], however efflux pump and ribosomal

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protection genes (tetM, tetO, etc.) are mostly fixed on smaller mobile genetic factors such as DNA sequences, transposons, and integrons, which more easily and freely pass through

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TRGs outside the cell [74, 75]. For these abovementioned reasons, tetA, tetC, tetO, and int1 showed higher penetration rates.

The relative abundance of TRGs and int1 (normalized to 16S rRNA) is shown in Fig.

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13b. Under all operating conditions, tetA and int1 together occupied a higher relative abundance in the NaCl solution, followed by tetC and tetO. The permeability of four

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different types of TRGs in different operating conditions were calculated to further analyze the interception effect of FO membranes on different functional TRGs. As illustrated in Fig. 13c, the efflux pump genes and ribosomal protection genes have the highest permeation rates. In FO mode, all groups of TRGs had lower permeation rates on the TFN2 membrane than other operating conditions. All of the abovementioned phenomena were consistent with conclusions shown in Fig. 13a, confirming that tetA, tetC, tetO, and int1 were more permeable across the membrane. Apart from that, all FO membranes showed superior 17

retention of enzymatic inactivation gene (tetX), and the permeability of tetX was less than 1%. Despite technical limitations, according to previous studies, FO membranes have been used in small-scale wastewater treatment plants for wastewater reuse [76-78]. Sanahuja-embuena et al. [79] investigated the performance of pilot-scale hollow fiber FO membranes under various operating conditions, FO had an increase rejection of 2% for caffeine, 19% for niacin, and 740% for urea compared to reverse osmosis membranes. Wang et al. [80] reported a pilot-scale FO system using a spiral-wound membrane module to

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concentrate real municipal wastewater, and the rejection rates of the system for COD and TP reached 99.8% and 99.7%, respectively. The system demonstrated the applicability of the FO process to wastewater concentration in wastewater treatment plants. Our experimental results show that the FO membrane and its modified membranes have a good retention effect on

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TRGs, which can provide a technical reference for the removal of multiple resistant genes

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and the treatment of antibiotics and resistance genes that co-exist in wastewater. 4. Conclusions

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TFC FO membranes with TiO2 and TiO2/AgNPs were successfully synthesized as demonstrated by SEM and FTIR analysis. According to the water contact angle test, the

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nanoparticle doping enhanced the hydrophilicity and improved the water flux. When treating the secondary effluent of the sewage treatment plant, the water flux of the FO membrane embedded in the TiO2/AgNPs attained 46.72 LMH and 56.49 LMH in two modes,

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respectively. In addition, compared with the TFN0 and TFN1 membranes, the TFN2 membrane exhibited strong fouling resistance and bacterial inhibition. All FO membranes

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effectively rejected TRB and TRGs. In particular, the permeability of the TFN2 membrane to TRB in the secondary effluent was as low as 0.75% due to the antibacterial activity verified by the diffusion inhibition zone test and cell viability tests. Furthermore, compared to the TRGs on the plasmids, the TRGs on the transposons (tetA, tetC, and tetO) and integrons (int1) were more permeable to membrane pores. Our results suggest that the FO membrane modified by antibacterial nanoparticles holds promise for the treatment of real wastewater 18

containing ARB and ARGs.

Declaration of interests 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.

Author contributions

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Haisheng Chen: Data curation, Formal analysis, Investigation, Roles/Writing - original draft, Methodology.

Manhong Huang: Conceptualization, Formal analysis, Funding acquisition, Resources, Supervision, Validation, Writing - review & editing.

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Shengyang Zheng: Formal analysis, Methodology.

Gang Chen: Writing - review & editing.

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Acknowledgments

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Xubiao Luo: Writing - review & editing.

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Lijun Meng: Formal analysis, Methodology.

The National Natural Science Foundation of China (No. 21477018), the fund from the Key Laboratory of Jiangxi Province for Persistent Pollutants Control and Resources Recycle

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(ES201980203), fundamental research funds for the central universities, and the National

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Key Research Development Program of China supported this work.

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Fig. 1. TEM images of the (a) TiO2 nanoparticles and (b) TiO2/AgNPs nanocomposite particles. (c) HRTEM of TiO2/AgNPs nanocomposite particles. Fig. 2. TEM micrographs of (a) PSf0 nanofiber. (b) PSf1 nanofiber. (c) PSf2 nanofiber. Element maps of (d) PSf1 nanofiber. (e) PSf2 nanofiber. Fig. 3. SEM images and fiber diameter distribution of different nanofiber substrates. Fig. 4. XRD spectra of PSf0, PSf1 and PSf2 nanofiber substrates. Fig. 5. (a) The top polyamide layer morphology, (b) sectional view and (c) the enlarged sectional view of the TFN2 membrane; (d) FTIR spectra of all kinds of FO membranes.

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Fig. 6. Water-contact angles of nanofiber substrates and FO membrane surfaces. Fig. 7. The antibacterial circle experiment of FO membranes in the disc diffusion test. Three parallel samples are prepared for all tests, TFN2 membranes showed obvious inhibition zones while other membrane samples showed no obvious phenomenon.

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Fig. 8. Bacterial morphology of E. coli cells under SEM on (a) TFN0, (b) TFN1 and (c) TFN2 membrane; (d) Normalized FO membrane inhibition rate by comparison with the

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number of viable bacteria on the TFN0 membrane.

Fig. 9. Process diagram of the TFN2 membrane to exert antibacterial effect.

lP

Figure 10. Filtration fouling test of the of TFN0, TFN1 and TFN2 membrane in FO mode: (a) multiple filtration cycle and cleaning experiment (0–60 min, water; 61–120 min,

na

secondary effluent; 121–180 min, water; 181–240 min, secondary effluent); (b) normalized water fluxes at 8h running time in biofouling experiments (the feed solution is 1L secondary effluent and the draw solution is 1M NaCl solution).

ur

Fig. 11. Water flux and TRB permeation rates by FO membranes in (a) FO mode and (b) PRO mode.

Jo

Fig. 12. Distribution of microorganisms in different water samples at phylum level. Fig. 13. (a) Permeation rates of TRGs, (b) the relative abundance of TRGs and int1 (normalized to 16S rRNA) in NaCl solution and (c) permeation rates of four types of TRGs in different operating conditions.

26

ro of

Fig. 1. TEM images of the (a) TiO2 nanoparticles and (b) TiO2/AgNPs nanocomposite

Jo

ur

na

lP

re

-p

particles. (c) HRTEM of TiO2/AgNPs nanocomposite particles.

Fig. 2. TEM micrographs of (a) PSf0 nanofiber. (b) PSf1 nanofiber. (c) PSf2 nanofiber. Element maps of (d) PSf1 nanofiber. (e) PSf2 nanofiber.

27

Average diameter:696.77 Error:88.51

PSf0

25

Count

20 15 10 5 0

500

600

700

800

900

1000

Fiber diameter(nm) Average diameter:895.63 Error:93.69

PSf1

25

Count

20 15 10 5 0

700

800

900

1000

1100

ro of

Fiber diameter(nm) 25

Average diameter:979.79 Error:119.31

PSf2

Count

20 15 10

0

-p

5

800

900

1000

1100

1200

Fiber diameter(nm)

（ 311）

（ 204） （ 220）

（ 105） （ 211）

re （ 200）

（ 004） （ 111）

（ 204）

（ 200）

（ 004）

（ 105） （ 211）

（ 101）

na PSf1

ur

Intensity (a. u.)

PSf2

（ 200）

lP

（ 101）

Fig. 3. SEM images and fiber diameter distribution of different nanofiber substrates.

Jo

PSf0

0

20

40 2q (degrees)

60

80

Fig. 4. XRD spectra of PSf0, PSf1 and PSf2 nanofiber substrates.

28

ro of -p

Fig. 5. (a) The top polyamide layer morphology, (b) cross-section microimage and (c) the

re

enlarged cross-section image of the TFN2 membrane; (d) FTIR spectra of all kinds of FO

lP

membranes.

140

100

80

ur

Contact angle (°)

na

120

PSf substrates FO mambranes

60

Jo

40 20

0

PSf0 TFN0

PSf1

TFN1

PSf2

TFN2

Membrane types

Fig. 6. Water-contact angles of nanofiber substrates and FO membrane surfaces.

29

Fig. 7. The antibacterial circle experiment of FO membranes in the disc diffusion test. Three parallel samples are prepared for all tests, TFN2 membranes showed obvious inhibition

ur

na

lP

re

-p

ro of

zones while other membrane samples showed no obvious phenomenon.

Fig. 8. Bacterial morphology of E. coli cells under SEM on (a) TFN0, (b) TFN1 and (c)

Jo

TFN2 membrane; (d) Normalized FO membrane inhibition rate by comparison with the number of viable bacteria on the TFN0 membrane.

30

ro of

（ a） Normalized water flux(Jw/Jw0)

0.9 0.7 0.6

（ b）

0.4 0.3 TFN0 TFN1 TFN2

0.2 0.1 0

50

0.8 0.6

lP

0.5

0.0

1.0

re

0.8

100 150 Time(min)

200

na

Normalized water flux(Jw/Jw0)

1.0

-p

Fig. 9. Process diagram of the TFN2 membrane to exert antibacterial effect.

250

0.4 TFN0 TFN1 TFN2

0.2 0.0

0

100

200 300 Time(min)

400

500

Fig. 10. Filtration fouling test of the of TFN0, TFN1 and TFN2 membrane in FO mode: (a)

ur

multiple filtration cycle and cleaning experiment (0–60 min, water; 61–120 min, secondary effluent; 121–180 min, water; 181–240 min, secondary effluent); (b) normalized water

Jo

fluxes at 8h running time in biofouling experiments (the feed solution is 1L secondary effluent and the draw solution is 1M NaCl solution).

31

70

50

1.5

60

1.8

40

1.2

50

1.5

40

1.2

30

0.9

30

0.9

20

0.6

20

0.6

10

0.3

10

0.3

0

TFN0

TFN2

TFN1

Water flux(LMH)

(a)

TRB permeability (%)

0.0

0

Jw

TFN0

Membrane types

(b)

TRB permeability (%)

TFN1

TFN2

2.1

TRB Permeation (%)

1.8

Jw

TRB Permeation (%)

Water flux(LMH)

60

0.0

Membrane types

Fig.

11. Water flux and TRB permeation rates by FO membranes in (a) FO mode and (b) PRO

ro of

mode.

-p

80

re

60

lP

40

20

na

Frequency of phylum (%)

100

0

Secondary effluent TFN0-FO

TFN0-PRO

TFN1-FO

TFN1-PRO

TFN2-FO

Unclassified Crenarchaeota Tenericutes Cloacimonetes Thermotogae Caldiserica candidate_ division_ WPS-1 Chlorobi BRC1 Microgenomates Fusobacteria SR1 Deinococcus -Thermus Latescibacteria Spirochaetes Gemmatimonadetes Hydrogenedentes Synergistetes Euryarchaeota Chlamydiae Ignavibacteriae Armatimonadetes Parcubacteria Nitrospirae Candidatus _ Saccharibacteria Firmicutes Verrucomicrobia Acidobacteria Atinobacteria Chlorofl exi Planctomycetes Bacteroidetes Proteobacteria

TFN2-PRO

ur

Water samples from different modes of operation

Jo

Fig. 12. Distribution of microorganisms in different water samples at phylum level.

32

0.01

（ c）

6 5

O N TF

N

2-

2PR

FO

O PR TF

N TF

Operating conditions

7

Efflux pump genes Ribosomal protection genes Enzymatic modification gene Integrase gene

3 2

PR O 2-

FO N

TF N

2-

PR TF

TF

N

1-

1N TF

TF

N

0P

0TF N

FO

O

FO

0

O

1

-p

Operating conditions

ro of

4

R

Permeation rates (%)

1-

N

Operating conditions

N

0FO

R O

TF

TF

TF N

N

1N TF

1E-6

2P

O

2FO

PR

O 1F TF N

0PR O N TF

TF N 0-

FO

0

1E-5

TF

1

FO

2

tetA tetC tetM tetO tetX int1

1E-4

1-

3

N

4

0.001

O

5

（ b）

PR

6

TF

tetA tetC tetM tetO tetX int1 16S rRNA

0-

（ a）

Gene copies/16S rRNA

Permeation rates (%)

7

Fig.

13. (a) Permeation rates of TRGs, (b) the relative abundance of TRGs and int1 (normalized

re

to 16S rRNA) in NaCl solution and (c) permeation rates of four types of TRGs in different

na

lP

operating conditions.

Fig. 1. TEM images of the (a) TiO2 nanoparticles and (b) TiO2/AgNPs nanocomposite

ur

particles. (c) HRTEM of TiO2/AgNPs nanocomposite particles. Fig. 2. TEM micrographs of (a) PSf0 nanofiber. (b) PSf1 nanofiber. (c) PSf2 nanofiber.

Jo

Element maps of (d) PSf1 nanofiber. (e) PSf2 nanofiber. Fig. 3. SEM images and fiber diameter distribution of different nanofiber substrates. Fig. 4. XRD spectra of PSf0, PSf1 and PSf2 nanofiber substrates. Fig. 5. (a) The top polyamide layer morphology, (b) sectional view and (c) the enlarged sectional view of the TFN2 membrane; (d) FTIR spectra of all kinds of FO membranes. Fig. 6. Water-contact angles of nanofiber substrates and FO membrane surfaces. Fig. 7. The antibacterial circle experiment of FO membranes in the disc diffusion test. Three 33

parallel samples are prepared for all tests, TFN2 membranes showed obvious inhibition zones while other membrane samples showed no obvious phenomenon. Fig. 8. Bacterial morphology of E. coli cells under SEM on (a) TFN0, (b) TFN1 and (c) TFN2 membrane; (d) Normalized FO membrane inhibition rate by comparison with the number of viable bacteria on the TFN0 membrane. Fig. 9. Process diagram of the TFN2 membrane to exert antibacterial effect. Figure 10. Filtration fouling test of the of TFN0, TFN1 and TFN2 membrane in FO mode: (a) multiple filtration cycle and cleaning experiment (0–60 min, water; 61–120 min,

ro of

secondary effluent; 121–180 min, water; 181–240 min, secondary effluent); (b) normalized water fluxes at 8h running time in biofouling experiments (the feed solution is 1L secondary effluent and the draw solution is 1M NaCl solution).

Fig. 11. Water flux and TRB permeation rates by FO membranes in (a) FO mode and (b)

-p

PRO mode.

Fig. 12. Distribution of microorganisms in different water samples at phylum level.

re

Fig. 13. (a) Permeation rates of TRGs, (b) the relative abundance of TRGs and int1 (normalized to 16S rRNA) in NaCl solution and (c) permeation rates of four types of TRGs

Jo

ur

na

lP

in different operating conditions.

34

ro of

Fig. 1. TEM images of the (a) TiO2 nanoparticles and (b) TiO2/AgNPs nanocomposite

Jo

ur

na

lP

re

-p

particles. (c) HRTEM of TiO2/AgNPs nanocomposite particles.

Fig. 2. TEM micrographs of (a) PSf0 nanofiber. (b) PSf1 nanofiber. (c) PSf2 nanofiber. Element maps of (d) PSf1 nanofiber. (e) PSf2 nanofiber.

35

Average diameter:696.77 Error:88.51

PSf0

25

Count

20 15 10 5 0

500

600

700

800

900

1000

Fiber diameter(nm) Average diameter:895.63 Error:93.69

PSf1

25

Count

20 15 10 5 0

700

800

900

1000

1100

ro of

Fiber diameter(nm) 25

Average diameter:979.79 Error:119.31

PSf2

Count

20 15 10

0

-p

5

800

900

1000

1100

1200

Fiber diameter(nm)

（ 311）

（ 204） （ 220）

（ 105） （ 211）

re （ 200）

（ 004） （ 111）

（ 204）

（ 200）

（ 004）

（ 105） （ 211）

（ 101）

na PSf1

ur

Intensity (a. u.)

PSf2

（ 200）

lP

（ 101）

Fig. 3. SEM images and fiber diameter distribution of different nanofiber substrates.

Jo

PSf0

0

20

40 2q (degrees)

60

80

Fig. 4. XRD spectra of PSf0, PSf1 and PSf2 nanofiber substrates.

36

ro of -p

Fig. 5. (a) The top polyamide layer morphology, (b) cross-section microimage and (c) the

re

enlarged cross-section image of the TFN2 membrane; (d) FTIR spectra of all kinds of FO

lP

membranes.

140

100

80

ur

Contact angle (°)

na

120

PSf substrates FO mambranes

60

Jo

40 20

0

PSf0 TFN0

PSf1

TFN1

PSf2

TFN2

Membrane types

Fig. 6. Water-contact angles of nanofiber substrates and FO membrane surfaces.

37

Fig. 7. The antibacterial circle experiment of FO membranes in the disc diffusion test. Three parallel samples are prepared for all tests, TFN2 membranes showed obvious inhibition

ur

na

lP

re

-p

ro of

zones while other membrane samples showed no obvious phenomenon.

Fig. 8. Bacterial morphology of E. coli cells under SEM on (a) TFN0, (b) TFN1 and (c)

Jo

TFN2 membrane; (d) Normalized FO membrane inhibition rate by comparison with the number of viable bacteria on the TFN0 membrane.

38

ro of

（ a） Normalized water flux(Jw/Jw0)

0.9 0.7 0.6

（ b）

0.4 0.3 TFN0 TFN1 TFN2

0.2 0.1 0

50

0.8 0.6

lP

0.5

0.0

1.0

re

0.8

100 150 Time(min)

200

na

Normalized water flux(Jw/Jw0)

1.0

-p

Fig. 9. Process diagram of the TFN2 membrane to exert antibacterial effect.

250

0.4 TFN0 TFN1 TFN2

0.2 0.0

0

100

200 300 Time(min)

400

500

Fig. 10. Filtration fouling test of the of TFN0, TFN1 and TFN2 membrane in FO mode: (a)

ur

multiple filtration cycle and cleaning experiment (0–60 min, water; 61–120 min, secondary effluent; 121–180 min, water; 181–240 min, secondary effluent); (b) normalized water

Jo

fluxes at 8h running time in biofouling experiments (the feed solution is 1L secondary effluent and the draw solution is 1M NaCl solution).

39

70

50

1.5

60

1.8

40

1.2

50

1.5

40

1.2

30

0.9

30

0.9

20

0.6

20

0.6

10

0.3

10

0.3

0

TFN0

TFN2

TFN1

Water flux(LMH)

(a)

TRB permeability (%)

0.0

0

Jw

TFN0

Membrane types

(b)

TRB permeability (%)

TFN1

TFN2

2.1

TRB Permeation (%)

1.8

Jw

TRB Permeation (%)

Water flux(LMH)

60

0.0

Membrane types

Fig.

11. Water flux and TRB permeation rates by FO membranes in (a) FO mode and (b) PRO

ro of

mode.

-p

80

re

60

lP

40

20

na

Frequency of phylum (%)

100

0

Secondary effluent TFN0-FO

TFN0-PRO

TFN1-FO

TFN1-PRO

TFN2-FO

Unclassified Crenarchaeota Tenericutes Cloacimonetes Thermotogae Caldiserica candidate_ division_ WPS-1 Chlorobi BRC1 Microgenomates Fusobacteria SR1 Deinococcus -Thermus Latescibacteria Spirochaetes Gemmatimonadetes Hydrogenedentes Synergistetes Euryarchaeota Chlamydiae Ignavibacteriae Armatimonadetes Parcubacteria Nitrospirae Candidatus _ Saccharibacteria Firmicutes Verrucomicrobia Acidobacteria Atinobacteria Chlorofl exi Planctomycetes Bacteroidetes Proteobacteria

TFN2-PRO

ur

Water samples from different modes of operation

Jo

Fig. 12. Distribution of microorganisms in different water samples at phylum level.

40

0.01

（ c）

6 5

O N TF

N

2-

2PR

FO

O PR TF

N TF

Operating conditions

7

Efflux pump genes Ribosomal protection genes Enzymatic modification gene Integrase gene

3 2

PR O 2-

FO N

TF N

2-

PR TF

TF

N

1-

1N TF

TF

N

0P

0TF N

FO

O

FO

0

O

1

-p

Operating conditions

ro of

4

R

Permeation rates (%)

1-

N

Operating conditions

N

0FO

R O

TF

TF

TF N

N

1N TF

1E-6

2P

O

2FO

PR

O 1F TF N

0PR O N TF

TF N 0-

FO

0

1E-5

TF

1

FO

2

tetA tetC tetM tetO tetX int1

1E-4

1-

3

N

4

0.001

O

5

（ b）

PR

6

TF

tetA tetC tetM tetO tetX int1 16S rRNA

0-

（ a）

Gene copies/16S rRNA

Permeation rates (%)

7

Fig.

13. (a) Permeation rates of TRGs, (b) the relative abundance of TRGs and int1 (normalized

re

to 16S rRNA) in NaCl solution and (c) permeation rates of four types of TRGs in different

Jo

ur

na

lP

operating conditions.

41

42

ro of

-p

re

lP

na

ur

Jo