Article pubs.acs.org/est
Cite This: Environ. Sci. Technol. 2018, 52, 8666−8673
Photocatalytic Reactive Ultrafiltration Membrane for Removal of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes from Wastewater Effluent Shaojie Ren,† Chanhee Boo,‡ Ning Guo,† Shuguang Wang,† Menachem Elimelech,‡ and Yunkun Wang*,†,‡ Environ. Sci. Technol. 2018.52:8666-8673. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 01/17/19. For personal use only.
†
Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Qingdao 266237, China ‡ Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States S Supporting Information *
ABSTRACT: Biological wastewater treatment is not effective in removal of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this study, we fabricated a photocatalytic reactive membrane by functionalizing polyvinylidene fluoride (PVDF) ultrafiltration (UF) membrane with titanium oxide (TiO2) nanoparticles for the removal of ARB and ARGs from a secondary wastewater effluent. The TiO2-modified PVDF membrane provided complete retention of ARB and effective photocatalytic degradation of ARGs and integrons. Specifically, the total removal efficiency of ARGs (i.e., plasmidmediated f loR, sul1, and sul2) with TiO2-modified PVDF membrane reached ∼98% after exposure to UV irradiation. Photocatalytic degradation of ARGs located in the genome was found to be more efficient than those located in plasmid. Excellent removal of integrons (i.e., intI1, intI2, and intI3) after UV treatment indicated that the horizontal transfer potential of ARGs was effectively controlled by the TiO2 photocatalytic reaction. We also evaluated the antifouling properties of the TiO2−UF membrane to demonstrate its potential application in wastewater treatment.
■
INTRODUCTION Widespread use of antibiotics results in the emergence of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in diverse environmental matrices, including soil, drinking water resources, and wastewater effluents. Stringent control of ARB and ARGs release to the environment is of critical importance because these microbial contaminants can potentially have a large negative impact on the environment and human health.1,2 Currently, most wastewater treatment plants (WWTPs) are not designed to eliminate microbial contaminants resistant to antibiotics, but rather become the main source for proliferation of ARB and ARGs due to the high concentration of activated sludge employed during biological wastewater treatment.3,4 Advanced oxidation processes such as chlorination, ozonation, and ultraviolet (UV) disinfection have recently been employed to control antibiotic resistant bacteria and genes from wastewater effluents. However, the effectiveness of these advanced oxidation treatment technologies for inactivation of ARB and ARGs is still controversial.5 For instance, Zhang et al. have demonstrated inactivation of tetracycline-resistant bacteria (TRB) containing tetC and tetA genes by sodium hypochlorite,6 whereas Munir et al. reported conflicting results indicating that chlorination is not effective in inactivating these ARB and ARGs.7 In another study, residual chlorine has been © 2018 American Chemical Society
shown to trigger the emergence of ARGs by acting like an antibiotic.8 Inactivation of ARB by ozone oxidation is possible, but the release of free ARGs from the bacteria cells decreases oxidation efficiency.5 UV treatment provides a promising solution for ARB removal by directly damaging the cell nucleic acid.9 However, employing high-intensity UV continuously during wastewater treatment remains a challenge.10−12 Moreover, the genes fragmented by oxidation can be integrated with other pathogens in the wastewater effluent, thereby requiring post-treatment.13,14 Thus, there is a critical need to develop alternative approaches for inactivation of ARB and further degradation of ARGs from wastewater effluents. Membrane technology has the potential to provide an effective solution for removal of ARB and ARGs from wastewater effluents.15−17 However, fouling by attachment of microbes on the membrane surface significantly deteriorates membrane performance.18,19 Furthermore, horizontal transfer of ARGs can be promoted within the fouling layer (i.e., biofilm) due to high bacteria density.20,21 Incorporating photocatalytic nanomaterials on the membrane surface can Received: Revised: Accepted: Published: 8666
April 10, 2018 July 5, 2018 July 9, 2018 July 9, 2018 DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology control biofouling during treatment of wastewater effluents contaminated by ARB and ARGs.22 Titanium dioxide (TiO2) provides a promising platform for the development of a photocatalytic reactive membrane, which enables inactivation of ARB and ARGs23,24 and reduces fouling potential through the generation of reactive oxygen species (ROS) under UV irradiation.25,26 The proliferation of ARGs is driven by vertical and horizontal gene transfers that are closely related to the types of bacteria host and mobilome, respectively.27 It is well-known that both genome and plasmid (i.e., mobile genetic element) in the bacteria cell contain ARGs and integrons.28,29 The ARGs carried by the genome are relatively stable in terms of their heredity. Thus, proliferation of these ARGs is mainly driven by vertical gene transfer. In contrast, horizontal transfer of ARGs takes place through transformation, transduction, or plasmid conjugation more easily and frequently. Integrons are considered an indicator of the potential for horizontal gene transfer due to their ability to capture exogenous gene cassettes.30,31 As with ARGs, the transfer mechanism of integrons is site-specific.32 For example, integrons located in plasmids have more chances for interspecies transmission, resulting in an increased ability to integrate with other bacteria. Likewise, degradation of integrons is expected to be dependent on their location in the bacteria cell, but studies of relevant mechanisms are still scarce. Therefore, fundamental understanding of the removal mechanisms of ARGs and integrons is critical for the development of effective control strategies. In this study, a photocatalytic reactive membrane was fabricated via a facile and scalable surface modification technique to remove antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from secondary wastewater effluent. A polyvinylidene fluoride (PVDF) ultrafiltration membrane was functionalized with TiO2 nanoparticles to enable photocatalytic degradation of ARB and ARGs. Removal efficiency and degradation mechanisms of ARB and ARGs were investigated. We also demonstrated the antifouling performance of the TiO2 functionalized PVDF membrane by evaluating fouling reversibility with secondary wastewater effluent. Implications of the results for the use photocatalytic reactive membranes to remove antibiotic resistant bacteria and genes in wastewater effluent are evaluated and discussed.
mg/mL dopamine solution in 10 mM Tris buffer (pH 8.5) was carefully poured onto the membrane surface. After 6 h, the polydopamine (PDA)-deposited membrane was removed from the frame and thoroughly rinsed with DI water three times. TiO2 (P25, 100 mg) was dispersed in 20 mL of DI water, followed by 10-min sonication in ultrasonic bath. The PDAcoated membrane was in contact with the TiO2 nanoparticle suspension (5 mg/mL) for 24 h. All surface modification steps were performed on a rocking platform at 30 °C. A schematic description of the PVDF membrane modification with TiO2 nanoparticles and the subsequent use of this membrane for ARB/ARGs filtration followed by UV photodegradation is presented in Supporting Information Figure S1. Membrane Characterization. Membrane morphology was characterized by scanning electron microscopy (SEM, Hitachi SU8010, Japan). Before imaging, samples were coated with a platinum layer to enhance membrane conductance. Membrane surface roughness was evaluated by atomic force microscopy (AFM, Dimension Icon, Veeco Instruments Inc., USA) in tapping mode. Membrane samples were dried in a vacuum desiccator for 24 h at 50 °C before AFM measurements. The water contact angle of the membranes was measured by a goniometer (Shanghai Zhongchen Digital Technology Apparatus Co., Ltd.) using the sessile drop method. A 2-μL water droplet was placed on the surface of the predried membrane and photographed using a digital camera. The measurements were conducted on a minimum of two random locations with three different membrane samples, and the data were averaged. Membrane porosity and water uptake were estimated gravimetrically.33 A 1.5 cm × 1.5 cm membrane sample was stored in DI water for 24 h and weighed carefully after removing excess water from the surface. Then, the sample was dried in a vacuum oven for 24 h and the weight was reevaluated. Porosity (ε) and water uptake values were calculated using
MATERIALS AND METHODS Materials and Chemicals. PVDF ultrafiltration membrane (Sepro, Carlsbad, CA) with a molecular weight cutoff of 100 kDa was used as a substrate. Before use, membranes were immersed in ethanol for 30 min, followed by three 30-min periods of rinse with deionized (DI) water to remove residual ethanol. Titanium oxide nanoparticles (TiO2, P25) were acquired from Evonik Industries AG. Tris-HCl solution (1 M, pH 9) and dopamine hydrochloride were purchased from Sinopharm Chemical Reagent Co., Ltd. The secondary wastewater effluent samples were collected from a WWTP located in Jinan City, China (36°42′11″N, 117°02′10″E). Functionalization of PVDF Membrane with TiO2. Prewetted PVDF membrane coupons were cut to fit a custom-made rectangular frame and held tightly by a soft rubber spacer using a clamp with the active surface layer facing up. A 10 mM Tris buffer was prepared by diluting the Tris-HCl solution (1 M, pH 9) in DI water and adjusting the pH to 8.5 using hydrochloric acid (HCl, 0.1 M). A freshly prepared 2
where Ww and Wd are the weights of the wet and dry membrane samples, respectively, ρw is the density of water, S is the membrane area, and l is the membrane thickness determined using a micrometer. Filtration and UV Photocatalytic Degradation Experiment. The separation and antifouling performance of the TiO2-modified PVDF membrane were evaluated in a dead-end UF system with a cell volume of 10 mL and an effective membrane area of 4.1 cm2 (Amicon stirred cell, model 8010), which was connected to a dispensing reservoir with a nitrogen gas supply. The secondary wastewater effluent was prefiltered through a filter paper (pore size of 80−120 μm) to remove large particulate and suspended matters. To determine the retention performance, 250 mL of secondary effluent was filtrated through the membrane at a pressure of 1.4 bar (20 psi). Then, 100 μL of sterile DI water was carefully dispensed on the membrane surface to wet the fouling layer, followed by membrane exposure to UV254 for 1 h. The distance between the membrane surface and UV lamp (QiPaiWells Company,
ji W − Wd zyz zz100 ε (%) = jjjj w z k ρw Sl {
ij W − Wd yz zz100 Water uptake (%) = jjj w j Wd zz k {
■
8667
(1)
(2)
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology
primers used in this work are listed in Table S1. The DNA of target gene was purified by the Qiagen Gel Extraction Kit (Qiagen, Germany) and ligated into a pMD 19-T vector (Takara, Japan) and cloned into E. coli DH5α competent cell (Takara, Japan). The transformed cells were grown in an LB selective medium containing 100 mg/L of ampicillin to determine the positive clones. A series of 10-fold dilutions of plasmid standards ranging from 109 to 104 copies μL−1 were conducted to generate a standard curve.7 Quantitative PCR (qPCR) was conducted in a 96-well plate containing 2 μL of plasmid or genomic DNA template, 10 μL of SYBR Premix Ex Taq (Takara, Japan), 0.4 μL of forward and reverse primers, and 7.2 μL of DNA-free water. All samples were run in triplicate in the qPCR reactions. Details on the qPCR measurements, including the sequences of ARGs and integrons, standard curves, and detection limits are presented in the Supporting Information (Table S2 and Table S3). Gene copy number was calculated from the corresponding standard curve. To clarify the removal mechanism, the Spearman correlation analysis between degradation efficiency of ARGs and integrons and a relative proportion of guanine and cytosine to the total nucleotides sequences (GC%) was performed using the Statistical Package for Social Science software (SPSS) based on a 5% significance level.
Shanghai) was maintained at 10 cm to obtain a consistent luminous intensity of 12 μW/cm2. Fouling reversibility was evaluated to investigate the antifouling property of the TiO2-modified PVDF membrane. Before fouling experiments, a new membrane coupon was compacted for 3 h under a pressure of 1.4 bar using DI water as the feed. After compaction, the membrane was further stabilized for 20 min to obtain a constant permeate water flux. Fouling experiments were conducted for 1 h using wastewater effluent as the feed at a pressure of 1.4 bar. After gentle rinsing with DI water, the fouled membrane was exposed to UV for 1 h. Last, the pure water flux was re-evaluated at 1.4 bar and the water flux recovery was calculated by comparing the value to that obtained with the fresh membrane coupon. After filtration and UV treatment, bacteria with intact and compromised cell membrane on the fouled membrane surface were quantified using confocal laser scanning microscopy (CLSM, FV1000-IX81, Olympus, Japan). The samples were stained with a solution of SYTO 9 and propidium iodide (PI) for 15 min in the dark, according to the reagent manual (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo Fisher Scientific, USA). After each staining step, the membrane samples were rinsed with sterile phosphate buffered saline (PBS, pH 7.2). Confocal images at random locations on the membrane surface were captured using a CLSM equipped with 20× flat field half apochromatic objective lens (UPLSAPO20). SYTO 9 was excited with an argon laser at 488 nm while PI was excited using a diode-pumped solid state (DPSS) 561 nm laser. Emissions were observed at 528 nm for live bacteria (stained with SYTO 9) and 645 nm for dead bacteria (stained with PI). SYTO 9 and PI were excited separately using two independent scans. FV10-ASW and Image-Pro Plus (version 6.0) software were used to analyze the confocal images. Plasmid and Genome DNA Extraction. For plasmid and genomic DNA extraction, each wastewater effluent sample (40 mL) collected before and after the UF experiments was centrifuged for 10 min at 10 000 rpm. Plasmid and genomic DNA were then extracted from the precipitates using the TIANpure Mini Plasmid Kit (TIANGEN) and TIANamp Bacteria DNA Kit (TIANGEN), respectively, following the instructions from the manufacturer. To extract plasmid and genomic DNA from the bacteria retained on the membrane surface, membrane coupons (4.1 cm2, before or after UV treatment) were immersed in 5 mL of sterile PBS and ultrasonicated for 20 min. Next, 2-mL aliquots of the ultrasonicated solution were placed in sterile centrifuge tubes for extracting the genome and plasmid DNA. All DNA samples were stored at −20 °C before use. The residual 1 mL of ultrasonicated solution was stored at 4 °C for bacteria count. Bacteria Count and qPCR Measurement. Bacteria abundance was determined by a plate count.34 Dilutions of the cell suspension were plated on Luria broth (LB, Thermo Fisher Scientific, USA) selective plates containing different antibiotics (chloramphenicol, tetracycline, and sulfadiazine) to select the bacteria resistant to the corresponding antibiotic. The concentrations of chloramphenicol, tetracycline, and sulfadiazine employed in the LB selective plates were 32, 16, and 512 mg/L, respectively.35 After incubation for 24 h at 37 °C, the ARB were quantified via a plate count. Quantitative PCR (qPCR) was performed to determine the abundances of various ARGs using a real-time PCR System (LightCycler480, Roche, Applied Science, Switzerland). The
■
RESULTS AND DISCUSSION Characteristics of TiO2-Modified PVDF Membrane. Surface morphology and structure of the TiO2-modified PVDF membrane were investigated by SEM. As shown in Figure 1a,
Figure 1. SEM and AFM micrographs of pristine PVDF membranes (a and d), PDA-coated membranes (b and e), and TiO2-modified (c and f) membranes. (g) Water contact angle and (h) porosity and water uptake of the various membranes. Error bars represent standard deviations from three independent replicates.
the pristine PVDF membrane has pores with sizes in the range of 20−50 nm in diameter. Surface morphology did not change much after a relatively long 6-h PDA coating as shown in Figure 1b. TiO2 nanoparticles were anchored to the PDAcoated PVDF membrane utilizing the catechol/quinone groups of PDA. Previous studies have reported that catechol/quinone groups enable homogeneous surface functionalization with TiO2 nanoparticles by preventing particle aggregation.36,37 We also evaluated the amount of TiO2 incorporated in the PVDF 8668
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology membranes using thermogravimetric analysis (TGA). Results from TGA indicate that TiO2 accounts for 1.5 wt % of the TiO2-modified PVDF membrane (Figure S2a). Images from SEM-EDS elemental mapping revealed evenly distributed TiO2 nanoparticles on the membrane surface (Figures S2b and S2c), consistent with SEM images of the surface of the TiO2modified PVDF membrane (Figure 1c). The average pore size of the PVDF membranes before and after surface modification was determined by the Brunauer− Emmett−Teller (BET) and Barrett−Joyner−Halenda (BJH) analyses using a gas sorptometer (SSA-4300, Builder, China) (Figure S3). We observed that surface modification with PDA and TiO2 reduced the average pore size of the PVDF membranes (pristine PVDF, 17 nm; PDA-coated PVDF, 15 nm; and TiO2-modified PVDF, 6 nm). AFM analysis indicates that coating of the PVDF membrane with PDA and with TiO2 nanoparticles had very little influence on the overall surface roughness (Figures 1d−f), although SEM images reveal a change in the overall surface morphology after TiO2 surface modification. Water contact angles for the pristine and surface-modified PVDF membranes were measured to evaluate the surface hydrophilicity. As shown in Figure 1g, the water contact angle of the PVDF membrane decreased after PDA modification, indicating that −OH and −NH2 functional groups of PDA render the membrane surface hydrophilic.38 Surface hydrophilicity was further increased after functionalization with TiO2 as evidenced by a water contact angle of the TiO2-modified PVDF membrane lower than that of the PDA-coated membrane, likely due to the hydroxyl groups of the TiO2 clusters.39 In contrast to surface hydrophilicity, the TiO2modified PVDF membrane exhibited the lowest water uptake. The observed lower water uptake of the TiO2-modified PVDF membrane compared to the pristine PVDF membrane is attributed to the reduction of membrane porosity (Figure 1h). Porosity translates to void volume within the membrane, which may impact water permeability and retention behaviors. The reduced pore size and more compact structure of the TiO2modified PVDF membrane compared to the pristine membrane is beneficial to the removal of bacteria as discussed in the following subsection. Removal and Inactivation of Total and Antibiotic Resistant Bacteria. Both membrane filtration (Figure 2a, left panel) and UV treatment (Figure 2a, right panel) provided, respectively, near complete removal and inactivation of total bacteria, especially with the TiO2-modified PVDF membrane. The TiO2-modified PVDF membrane exhibited over 99.9% retention of total bacteria, higher than the pristine PVDF membrane (98.9%). The higher total bacteria removal efficiency of the TiO2-modified PVDF membrane compared to that of the pristine PVDF membrane was further confirmed by the reduced abundance of 16S rRNA in the permeate (Figure S4a). It is noteworthy that a small amount of bacteria passed through both the pristine and TiO2-modified PVDF membranes, although bacteria (size of 0.1−2 μm, refer to inset of Figure 2b) are larger than the size of membrane pores (several tens-of-nanometer, Figures 1a and 1c and Figure S3). Previous studies have reported that bacteria permeation through microporous membranes depends on the structural properties of bacteria, including size and shape,40,41 and hydrodynamic filtration conditions.42 The incomplete removal of bacteria by the investigated UF membranes is likely because bacteria deformed under the hydraulic pressure (1.4 bar) pass
Figure 2. (a, left) Total bacteria abundance in the feed and filtrate obtained from UF experiments. The secondary wastewater effluent was filtered by the pristine PVDF and TiO2-modified PVDF membranes until the permeate volume reached 250 mL, at a pressure of 1.4 bar (20 psi) and temperature of 25.0 ± 0.5 °C. (a, right) Photocatalytic degradation of total bacteria on the surface of the pristine PVDF and TiO2-modified PVDF membranes before and after exposure to UV for 1 h. (b) CFU of antibiotic resistant bacteria (ARB) on the surfaces of the pristine PVDF membrane and TiO2modified PVDF membrane, respectively, before and after exposure to UV irradiation, measured via spread plate method. Microscopic images of ARB are shown in the inset with a 2-μm scale bar.
through the membrane despite their much larger intrinsic size than membrane pores.41 The TiO2-modified PVDF membrane exhibited much higher retention of tetracycline-resistant bacteria (TRB) and chloramphenicol-resistant bacteria (CRB) than the pristine PVDF membrane, while the retention of sulfadiazine-resistant bacteria (SRB) displayed an opposite result. To explain the observed different ARB retention behaviors, ARB from the wastewater sample were isolated and examined by optical microscopy (insets of Figure 2b). Results indicate that TRB and CRB are mostly coccus with a spherical or round shape and much smaller size compared to SRB, which is a rod-shaped bacillus. Such structural properties of TRB and CRB result in a lower retention by the pristine PVDF membrane compared to that by the TiO2-modified PVDF membrane which has smaller pores than the pristine PVDF membrane (Figure 1c). It can be inferred from this observation that TRB and CRB in wastewater effluent are more challenging to remove than SRB by the UF membrane. The TiO2-modified PVDF membrane showed significant improvement in inactivation of total bacteria after UV treatment compared with the pristine PVDF membrane (Figure 2a, right panel). Similarly, the TiO2-modified PVDF membrane exhibited a higher photocatalytic degradation 8669
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology efficiency of ARB than the pristine PVDF membrane (Figure 2b). To better evaluate the effect of TiO2 photocatalytic reaction on bacteria inactivation, the live/dead bacteria retained on the membrane surface were quantified via CLSM analysis. As Figure 3 shows, more dense population of dead
Figure 4. Retention of ARGs and integrons by the pristine PVDF and TiO2-modified PVDF membranes. The UF experiments were conducted at 1.4 bar (20 psi) until cumulative permeate volume reached 250 mL. The feed and permeate were collected and analyzed for abundance of ARGs and integrons that position in genome (a, b) and plasmid (c, d).
genes were not detectable in the genome, indicating that tetQ gene transfer was mainly carried through the plasmid (Figures 4a and 4c). This result also implies that ARGs have distinctive locational distribution in the microorganisms which exist in the effluent of wastewater treatment plants. All three integrons (intI1, intI2, and intI3) were found in the secondary wastewater effluent. Specifically, intI1 accounted for the major portion (over 103 copies/mL in genome and plasmid), while intI2 was detected at very low concentration (less than 5 copies/mL) in both genome and plasmid (Figures 4b and 4d). As shown in Figure S5a−d, removal efficiencies of ARGs and integrons by the pristine and TiO2-modified PVDF membranes differ substantially, depending on their locations in the bacteria cell. Genome size is reported to be in the range of ∼130 kbp to ∼14 Mbp, while plasmid has relatively small sizes ranging from ∼1 to ∼100 kbp.43,44 Thus, ARGs located in the genome were better retained by the pristine and TiO2-modified PVDF membranes than those located in the plasmid (Figures 4a and 4c). The ARGs carried by the genome are more stable in terms of their heredity than those carried by plasmid, and thus propagate mainly through vertical gene transfer.45 A relatively high removal efficiency of ARGs in the genome by both the pristine and TiO2-modified PVDF membranes indicates that the potential of vertical ARG transfer can effectively be controlled by membrane filtration. We also found that removal of integrons in the genome was more efficient than those in plasmid by the pristine and TiO2-modified PVDF membranes (Figures 4b and 4d). Degradation of Retained Antibiotic Resistance Genes and Integrons. To determine the photocatalytic degradation of ARGs and integrons retained by the membrane, we exposed the membrane surface after filtration experiments to UV irradiation for 1 h. As shown in Figure 5, ARGs and integrons located in the genome had a higher photocatalytic degradation efficiency compared to those in plasmid. This observation indicates that degradation of ARGs and integrons by UV photocatalysis depends on their location in the bacteria cell. Compared with the relatively small size plasmid, the larger genome can easily be attacked by reactive oxygen species
Figure 3. CLSM images representing intact bacteria (green) and damaged bacteria (red) on (a) pristine PVDF, (b) pristine PVDF after UV treatment, (c) TiO2-modified PVDF, and (d) TiO2-modified PVDF membranes after UV treatment.
cells (shown red) was observed on the TiO2-modified PVDF membrane (Figures 3c and 3d) compared to that on the pristine PVDF membrane (Figures 3a and 3b) after UV treatment. We further verified the strong photocatalytic degradation efficiency of TiO2 by the reduced abundance of the 16S rRNA genes in the permeate obtained from UF experiments with the TiO2-modified PVDF membrane compared to that obtained with the pristine PVDF membrane (Figure S4b). It is interesting to note that some bacteria are destroyed during filtration by the TiO2-modified PVDF membrane (compare Figures 3a and 3c). In contrast, photocatalytic inactivation of bacteria retained on the pristine PVDF membrane was incomplete after UV treatment (Figures 3b and 3d), suggesting that surface functionalization with TiO2 is critical to achieve high inactivation rate of bacteria. Retention of Antibiotic Resistance Genes and Integrons. The abundance of chloramphenicol resistance gene (f loR), tetracycline resistance genes (tetC, tetW, and tetQ), sulfonamides resistance genes (sul1 and sul2), and three integrons in the feed (i.e., secondary wastewater effluent) and in the permeate measured after filtration with the pristine and TiO2-modified PVDF membranes is presented in Figure 4. Compared with the pristine PVDF membrane, the TiO2modified membrane showed increased retention of most ARGs and integrons because the attached TiO2 nanoparticles form a denser membrane structure. Notably, approximately several tens to hundreds of copies of tetQ gene in plasmid were measured in the feed and permeate obtained with both the pristine and TiO2-modified PVDF membranes, while these 8670
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology
resistance genes located in both the genome and plasmid by UV irradiation. Antifouling Performance of TiO2-Modified PVDF Membrane. To investigate the antifouling performance of the TiO2-modified membrane, we conducted dead-end UF experiments to evaluate water flux recovery after fouling. Rapid water flux decline was observed for both the pristine and TiO2modified PVDF membranes with a secondary wastewater effluent feed solution (Figure 6). A substantial fouling layer is
Figure 5. Photocatalytic degradation of ARGs and integrons on the surface of pristine PVDF and TiO2-modified PVDF membranes after UV treatment. ARGs and integrons in genome (a, b) and plasmid (c, d) were extracted using bacteria DNA kit and plasmid kit, respectively, and analyzed via quantitative PCR method.
Figure 6. Antifouling performance of the pristine PVDF and TiO2modified PVDF membranes evaluated using a dead-end UF setup. Before fouling runs, membranes were compacted for 3 h at 1.4 bar (20 psi) until a steady flux was obtained. Fouling experiments were conducted for 60 min using pretreated (by a filter paper with a pore size of 80−120 μm) secondary effluent from a wastewater treatment plant as a feed at applied pressure of 1.4 bar (20 psi) and temperature of 25.0 ± 0.5 °C. Initial water fluxes of the pristine PVDF and TiO2modified PVDF membrane were 1225 and 1925 L m−2h−1, respectively. After fouling experiments, fouled membranes were exposed to UV irradiation for 1 h and pure water flux was reevaluated at an applied pressure of 1.4 bar (20 psi).
(ROS) produced by TiO2 under UV irradiation. The TiO2modified PVDF membrane showed higher degradation efficiencies for most ARGs and integrons located in both the genome and plasmid than the pristine PVDF membrane after UV treatment (Figures 5a−d), in accordance with the previous results on total and antibiotic resistant bacteria inactivation (shown earlier in Figure 2). The degradation efficiencies of f loR, tetC, sul1, and intI1 in the plasmid by the TiO2-modified PVDF membrane were 97.82%, 20.66%, 99.45%, and 93.67%, respectively, which are higher than those by the pristine PVDF membrane (93.07%, 15.06%, 99.29%, and 88.86%, respectively) after UV treatment. Although these improvements were not substantial (P-value of 0.001, 0.009, 0.116, and 0.021 for the f loR, tetC, sul1, and intI1, respectively), the results indicate that the degradation of ARGs and integrons was enhanced by the photocatalytic activity of TiO2. Photocatalytic degradation efficiency of tetracycline resistance genes (tetC, tetW, and tetQ) was lower than that of sulfonamides resistance genes (sul1 and sul2) located in both the plasmid and genome. The degradation efficiencies of tetC, tetW, and tetQ in the plasmid were only 20.6%, 27.2%, and 2.0%, respectively, for the TiO2-modified PVDF membrane after UV treatment, which is much lower than those of sul1 (99.3%) and sul2 (98.8%). This finding is in good agreement with results from previous studies showing that tetracycline resistance genes are more difficult to degrade by UV disinfection than sulfonamides resistance genes (Figure 5c).46 Oxidants generated by an indirect photolysis pathway, such as reactive oxygen species (ROS), preferentially react with guanine bases in DNA.47,48 We found a strong correlation between the guanine-cytosine content (or GC%) and DNA degradation efficiency of the investigated ARGs and integrons by UV treatment; this finding indicates that ROS generated via TiO2 photocatalytic reaction was the major contributor for degradation of ARGs and integrons located in both the genome and plasmid (Table S4). The GC% of tetracycline resistance genes, especially tetQ (46%), was lower than that of sulfonamides resistance genes (Table S2), which explains the previously observed lower degradation efficiency of tetracycline
likely to form on the membrane surface due to the high foulant loading in the wastewater effluent, causing a sharp decrease of water flux. After the fouling experiments, membranes were exposed to UV irradiation for 1 h to degrade ARB and ARGs retained on the membrane surface. We observed a much higher flux recovery for the TiO2-modified PVDF membrane compared to the pristine PVDF membrane after UV treatment. Furthermore, long-term UF operation with several filtration cycles indicated that the recovery of water flux for the TiO2modified PVDF membrane remained high and exceeded significantly that of the pristine PVDF membrane (Figure S6). The stability of the TiO2 surface coating during filtration was evaluated by analyzing the titanium concentration in the permeate after UF experiments. Even after 48-h long-term UF experiments, titanium concentration in the permeate was only ∼0.3 ng/L (Figure S7), substantially lower compared to the amount of TiO2 deposited on the membrane. Hence, the amount of TiO2 leaching from the membrane surface during filtration is negligible and will not compromise membrane performance and permeate water quality. The observed antifouling property of the TiO2-modified PVDF membrane is attributed to the ROS produced by the TiO2 nanoparticles under UV irradiation, which effectively inactivate the ARB and ARGs, thereby disrupting the fouling layer. Previous studies have reported that the PDA intermediate layer could act as a free-radical scavenger to prevent the substrate from damage by UV irradiation.49 Thus, the PDA layer coated on the PVDF substrate also contributes to the almost complete water flux recovery of the TiO2modified membrane after UV treatment. The observed 8671
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology
(4) Li, N.; Sheng, G. P.; Lu, Y. Z.; Zeng, R. J.; Yu, H. Q. Removal of antibiotic resistance genes from wastewater treatment plant effluent by coagulation. Water Res. 2017, 111, 204−212. (5) Zheng, J.; Su, C.; Zhou, J. W.; Xu, L. K.; Qian, Y. Y.; Chen, H. Effects and mechanisms of ultraviolet, chlorination, and ozone disinfection on antibiotic resistance genes in secondary effluents of municipal wastewater treatment plants. Chem. Eng. J. 2017, 317, 309− 316. (6) Zhang, T.; Zhang, M.; Zhang, X. X.; Fang, H. H. Tetracycline resistance genes and tetracycline resistant lactose-fermenting Enterobacteriaceae in activated sludge of sewage treatment plants. Environ. Sci. Technol. 2009, 43 (10), 3455−3460. (7) Munir, M.; Wong, K.; Xagoraraki, I. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan. Water Res. 2011, 45 (2), 681−693. (8) Li, D.; Zeng, S. Y.; He, M.; Gu, A. Z. Water disinfection byproducts induce antibiotic resistance-role of environmental pollutants in resistance phenomena. Environ. Sci. Technol. 2016, 50 (6), 3193−3201. (9) Sinha, R. P.; Hader, D. P. UV-induced DNA damage and repair: a review. Photochem. Photobiol. Sci. 2002, 1 (4), 225−236. (10) Chen, H.; Zhang, M. M. Effects of advanced treatment systems on the removal of antibiotic resistance genes in wastewater treatment plants from Hangzhou, China. Environ. Sci. Technol. 2013, 47 (15), 8157−8163. (11) Mauter, M. S.; Zucker, I.; Perreault, F.; Werber, J. R.; Kim, J. H.; Elimelech, M. The role of nanotechnology in tackling global water challenges. Nat. Sustain. 2018, 1 (4), 166−175. (12) Lee, J.; Jeon, J. H.; Shin, J.; Jang, H. M.; Kim, S.; Song, M. S.; Kim, Y. M. Quantitative and qualitative changes in antibiotic resistance genes after passing through treatment processes in municipal wastewater treatment plants. Sci. Total Environ. 2017, 605, 906−914. (13) de Vries, J.; Wackernagel, W. Integration of foreign DNA during natural transformation of Acinetobacter sp by homologyfacilitated illegitimate recombination. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (4), 2094−2099. (14) Nielsen, K. M.; Johnsen, P. J.; Bensasson, D.; Daffonchio, D. Release and persistence of extracellular DNA in the environment. Environ. Biosaf. Res. 2007, 6 (1−2), 37−53. (15) Riquelme Breazeal, M. V.; Novak, J. T.; Vikesland, P. J.; Pruden, A. Effect of wastewater colloids on membrane removal of antibiotic resistance genes. Water Res. 2013, 47 (1), 130−140. (16) Cheng, H.; Hong, P. Y. Removal of antibiotic-resistant bacteria and antibiotic resistance genes affected by varying degrees of fouling on anaerobic microfiltration membranes. Environ. Sci. Technol. 2017, 51 (21), 12200−12209. (17) Zhu, Y. J.; Wang, Y. Y.; Zhou, S.; Jiang, X. X.; Ma, X.; Liu, C. Robust performance of a membrane bioreactor for removing antibiotic resistance genes exposed to antibiotics: role of membrane foulants. Water Res. 2018, 130, 139−150. (18) Tian, J. Y.; Ernst, M.; Cui, F. Y.; Jekel, M. Correlations of relevant membrane foulants with UF membrane fouling in different waters. Water Res. 2013, 47 (3), 1218−1228. (19) Haberkamp, J.; Ernst, M.; Böckelmann, U.; Szewzyk, U.; Jekel, M. Complexity of ultrafiltration membrane fouling caused by macromolecular dissolved organic compounds in secondary effluents. Water Res. 2008, 42 (12), 3153−3161. (20) Balcázar, J. L.; Subirats, J.; Borrego, C. M. The role of biofilms as environmental reservoirs of antibiotic resistance. Front. Microbiol. 2015, 6, 1216. (21) Á guila-Arcos, S.; Á lvarez-Rodríguez, I.; Garaiyurrebaso, O.; Garbisu, C.; Grohmann, E.; Alkorta, I. Biofilm-forming clinical Staphylococcus isolates harbor horizontal transfer and antibiotic resistance genes. Front. Microbiol. 2017, 8, 2018. (22) Pan, H.; Jian, Y. F.; Chen, C. W.; He, C.; Hao, Z. P.; Shen, Z. X.; Liu, H. X. Sphere-shaped Mn3O4 catalyst with remarkable lowtemperature activity for methyl-ethyl-ketone combustion. Environ. Sci. Technol. 2017, 51 (11), 6288−6297.
membrane stability and antifouling performance of the TiO2modified PVDF membrane with UV treatment demonstrate the potential application of photocatalytic reactive membranes in treatment of wastewater effluents contaminated by ARB and ARGs.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.8b01888. Scheme illustrating the functionalization of PVDF membrane and removal/degradation mechanisms of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) (Figure S1); Amount and areal distribution of TiO2 incorporated on the PVDF membrane (Figure S2); Nitrogen adsorption−desorption isotherms and pore size distribution of membranes (Figure S3); Abundance of 16S rRNA in the feed, permeate, and from bacteria retained on the membrane (Figure S4); Removal efficiencies of ARGs and integrons by UF (Figure S5); Antifouling performance in longterm UF operation (Figure S6); Amount of TiO2 leaching during filtration (Figure S7); Information on primers used for qPCR (Table S1); Annealing temperatures and sequences of ARGs (Table S2); Standard curves and LOD95 of ARGs and integrons (Table S3); Spearman’s correlation coefficients between degradation efficiency of ARGs and integrons and a proportion of guanine and cytosine to the total nucleotides sequences (GC%) (Table S4) (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; phone: +86 (532) 58630960. ORCID
Chanhee Boo: 0000-0003-4595-9963 Menachem Elimelech: 0000-0003-4186-1563 Yunkun Wang: 0000-0003-2848-3633 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We acknowledge the support received from the NSFC (51508309), China Postdoctoral Science Foundation (2015M570596 and 2017T100496), and the Research Award Fund for Outstanding Young Scientists of Shandong Province (BS2015HZ013).
■
REFERENCES
(1) Zhu, Y. G.; Zhao, Y.; Li, B.; Huang, C. L.; Zhang, S. Y.; Yu, S.; Chen, Y. S.; Zhang, T.; Gillings, M. R.; Su, J. Q. Continental-scale pollution of estuaries with antibiotic resistance genes. Nat. Microbiol. 2017, 2, 16270. (2) Martinez, J. L. Antibiotics and antibiotic resistance genes in natural environments. Science 2008, 321 (5887), 365−367. (3) Calero-Caceres, W.; Melgarejo, A.; Colomer-Lluch, M.; Stoll, C.; Lucena, F.; Jofre, J.; Muniesa, M. Sludge as a potential important source of antibiotic resistance genes in both the bacterial and bacteriophage fractions. Environ. Sci. Technol. 2014, 48 (13), 7602− 7611. 8672
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
Article
Environmental Science & Technology (23) Rizzo, L.; Sannino, D.; Vaiano, V.; Sacco, O.; Scarpa, A.; Pietrogiacomi, D. Effect of solar simulated N-doped TiO2 photocatalysis on the inactivation and antibiotic resistance of an E. coli strain in biologically treated urban wastewater. Appl. Catal., B 2014, 144, 369−378. (24) Ferro, G.; Fiorentino, A.; Alferez, M. C.; Polo-López, M. I.; Rizzo, L.; Fernández-Ibáñez, P. Urban wastewater disinfection for agricultural reuse: effect of solar driven AOPs in the inactivation of a multidrug resistant E. coli strain. Appl. Catal., B 2015, 178, 65−73. (25) Geng, Z.; Yang, X.; Boo, C.; Zhu, S. Y.; Lu, Y.; Fan, W.; Huo, M. X.; Elimelech, M.; Yang, X. Self-cleaning anti-fouling hybrid ultrafiltration membranes via side chain grafting of poly(aryl ether sulfone) and titanium dioxide. J. Membr. Sci. 2017, 529, 1−10. (26) Petosa, A. R.; Brennan, S. J.; Rajput, F.; Tufenkji, N. Transport of two metal oxide nanoparticles in saturated granular porous media: role of water chemistry and particle coating. Water Res. 2012, 46 (4), 1273−1285. (27) Dobrindt, U.; Hochhut, B.; Hentschel, U.; Hacker, J. Genomic islands in pathogenic and environmental microorganisms. Nat. Rev. Microbiol. 2004, 2, 414. (28) Ma, L.; Li, A. D.; Yin, X. L.; Zhang, T. The prevalence of integrons as the carrier of antibiotic resistance genes in natural and man-made environments. Environ. Sci. Technol. 2017, 51 (10), 5721− 5728. (29) Zhou, W. Q.; Yao, K. H.; Zhang, G.; Yang, Y. H.; Li, Y.; Lv, Y.; Feng, J. Mechanism for transfer of transposon Tn2010 carrying macrolide resistance genes in Streptococcus pneumoniae and its effects on genome evolution. J. Antimicrob. Chemother. 2014, 69 (6), 1470− 1473. (30) Hu, H. W.; Wang, J. T.; Li, J.; Shi, X. Z.; Ma, Y. B.; Chen, D.; He, J. Z. Long-term nickel contamination increases the occurrence of antibiotic resistance genes in agricultural soils. Environ. Sci. Technol. 2017, 51 (2), 790−800. (31) Gao, P.; Gu, C.; Wei, X.; Li, X.; Chen, H.; Jia, H.; Liu, Z.; Xue, G.; Ma, C. The role of zero valent iron on the fate of tetracycline resistance genes and class 1 integrons during thermophilic anaerobic co-digestion of waste sludge and kitchen waste. Water Res. 2017, 111, 92−99. (32) Mazel, D. Integrons: agents of bacterial evolution. Nat. Rev. Microbiol. 2006, 4, 608. (33) Demirel, E.; Zhang, B. P.; Papakyriakou, M.; Xia, S. M.; Chen, Y. S. Fe2O3 nanocomposite PVC membrane with enhanced properties and separation performance. J. Membr. Sci. 2017, 529, 170−184. (34) Pei, R.; Kim, S.-C.; Carlson, K. H.; Pruden, A. Effect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Res. 2006, 40 (12), 2427−2435. (35) Guo, M. T.; Yuan, Q. B.; Yang, J. Microbial selectivity of UV treatment on antibiotic-resistant heterotrophic bacteria in secondary effluents of a municipal wastewater treatment plant. Water Res. 2013, 47 (16), 6388−6394. (36) Zhang, R. X.; Braeken, L.; Luis, P.; Wang, X. L.; Van der Bruggen, B. Novel binding procedure of TiO2 nanoparticles to thin film composite membranes via self-polymerized polydopamine. J. Membr. Sci. 2013, 437, 179−188. (37) Wu, H. Q.; Liu, Y. J.; Mao, L.; Jiang, C. H.; Ang, J. M.; Lu, X. H. Doping polysulfone ultrafiltration membrane with TiO2-PDA nanohybrid for simultaneous self-cleaning and self-protection. J. Membr. Sci. 2017, 532, 20−29. (38) Liu, Y. L.; Ai, K. L.; Lu, L. H. Polydopamine and its derivative materials: synthesis and promising applications in energy, environmental, and biomedical fields. Chem. Rev. 2014, 114 (9), 5057−5115. (39) Xu, Z. W.; Wu, T. F.; Shi, J.; Teng, K. Y.; Wang, W.; Ma, M. J.; Li, J.; Qian, X. M.; Li, C. Y.; Fan, J. T. Photocatalytic antifouling PVDF ultrafiltration membranes based on synergy of graphene oxide and TiO2 for water treatment. J. Membr. Sci. 2016, 520, 281−293. (40) Foley, G. A review of factors affecting filter cake properties in dead-end microfiltration of microbial suspensions. J. Membr. Sci. 2006, 274 (1−2), 38−46.
(41) Lebleu, N.; Roques, C.; Aimar, P.; Causserand, C. Role of the cell-wall structure in the retention of bacteria by microfiltration membranes. J. Membr. Sci. 2009, 326 (1), 178−185. (42) Gaveau, A.; Coetsier, C.; Roques, C.; Bacchin, P.; Dague, E.; Causserand, C. Bacteria transfer by deformation through microfiltration membrane. J. Membr. Sci. 2017, 523, 446−455. (43) Bennett, G. M.; Moran, N. A. Small, smaller, smallest: the origins and evolution of ancient dual symbioses in a phloem-feeding insect. Genome Biol. Evol. 2013, 5 (9), 1675−1688. (44) Slater, F. R.; Bailey, M. J.; Tett, A. J.; Turner, S. L. Progress towards understanding the fate of plasmids in bacterial communities. FEMS Microbiol. Ecol. 2008, 66 (1), 3−13. (45) Summers, D. K.; Sherratt, D. J. Multimerization of high copy number plasmids causes instability: ColE 1 encodes a determinant essential for plasmid monomerization and stability. Cell 1984, 36 (4), 1097−1103. (46) Auerbach, E. A.; Seyfried, E. E.; McMahon, K. D. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res. 2007, 41 (5), 1143−51. (47) Qiao, Z.; Wigginton, K. R. Direct and indirect photochemical reactions in viral RNA measured with RT-qPCR and mass spectrometry. Environ. Sci. Technol. 2016, 50 (24), 13371−13379. (48) Cadet, J.; Douki, T., Formation of UV-induced DNA damage contributing to skin cancer development. Photochem. Photobiol. Sci. 2018. DOI: 10.1039/C7PP00395A (49) Feng, K.; Hou, L.; Tang, B. B.; Wu, P. Y. A self-protected selfcleaning ultrafiltration membrane by using polydopamine as a freeradical scavenger. J. Membr. Sci. 2015, 490, 120−128.
8673
DOI: 10.1021/acs.est.8b01888 Environ. Sci. Technol. 2018, 52, 8666−8673
